Methods, compositions and kits for vegetative cell-based vaccines and spore-based vaccines

ABSTRACT

Methods for immunizing a subject to an antigen of an infectious agent, a tumor, or an allergen are provided, using vegetative cytoplasmic expression of the antigen or spore surface display of the antigen, and contacting the subject with a composition including a spore or a vegetative cell or both with or without an adjuvant. Also included are thermally-stable vaccine compositions using the method described above and kits including the compositions.

RELATED APPLICATIONS

The present invention claims the benefit of and is a continuation inpart of PCT application number PCT/US2009/50356 filed Jul. 13, 2009which claims the benefit of U.S. provisional application Ser. No.61/134,700 filed Jul. 11, 2008, and also claims the benefit of U.S.provisional application Ser. No. 61/405,950 filed Oct. 22, 2010, each ofwhich is hereby incorporated by reference herein in its entirety.

TECHNICAL FIELD

Methods and vaccines are provided for immunizing a subject with avaccine that is a fusion to a spore coat protein or a vegetativecytoplasmic expression of an antigen of an infectious agent, a tumor, oran allergen and thermally-stable vaccine compositions.

BACKGROUND

Bacillus subtilis is a Gram-positive, catalase-positive bacteriumcommonly found in soil. Members of the genus Bacillus have the abilityto form tough, protective endospores, a characteristic which allows thespores of the organism to tolerate extreme environmental conditions, tobe heat resistant, and to quantitatively survive lengthy exposure to awide range of temperatures including freezing and boiling, without lossof viability.

B. subtilis has a long safety record as a food component and as aprobiotic, e.g., used in microbial feed supplements to improveintestinal microbial balance by competitively excluding pathogens bothin animals and humans. Other Bacillus species are well-known biologicalinsecticidal agents, e.g., Bacillus thuringiensis (Dipel®) is used tocombat gypsy moths without harm to other wildlife. Bacillus nattoincludes food-grade strains mainly used for the fermentation ofsoybeans, which fermentation process eventually results in a cheap andnutritious food that is rich in amino acids. In fact the term “natto”refers to a Japanese soybean fermented product “Natto”, which is awidely used commercial product.

Bacilli have been studied extensively by researchers and as a resultthis family includes species with well-characterized genetic andphysiological systems. B. subtilis has become a model organism forGram-positive bacteria, and numerous studies have been publishedinvolving manipulation of its genetic structure and regulation ofexpression of its proteins.

There remains a need for vaccines that are easily produced in largequantities and at low cost to prevent and control emerging viralepidemic and epizootic diseases. Vaccines based on bacterial productionsystems that can be stabilized for use in tropical areas and under otherconditions to minimize loss of activity in areas having minimal storagecapabilities are available.

SUMMARY

An embodiment of the invention provides a method of immunizing a subjectto an infectious agent, a tumor, or an allergen, the method including:providing a vegetative host bacterial cell including an isolatednucleotide sequence encoding an antigen of the infectious agent, thetumor, or the allergen, such that the nucleotide sequence is operablylinked to a promoter for cytoplasmic vegetative expression of theantigen or for expression of the antigen as a fusion to a spore coatprotein, such that at least one of vegetative cells and spores areassociated with the antigen; and, contacting a mucosal tissue of thesubject with a composition including at least one of the vegetativecells and the spores, such that the antigen immunizes the subject to theinfectious agent, the tumor, or to the allergen.

In a related embodiment of the method, the infectious agent is selectedfrom: a bacterium, a fungus, a virus, a protozoan, or a protein productthereof. In a related embodiment, the infectious agent is at least onebacterium selected from: Bacillus for example B. anthracis; Clostridiumfor example C. tetani, C. difficile, and C. perfringens; Corynebacteriumfor example C. diphtherias; Bordetella for example B. pertussis;Mycobacterium for example M. tuberculosis; Salmonella for example S.enterica; Staphylococcus for example S. aureus and S. epidermis;Streptococcus for example S. pneumoniae and S. mutans; Treponema forexample T. pallidum; Plasmodium for example P. falciparum, P. vivax, P.malariae, and P. ovale; Pseudomonas for example P. aeruginosa; Neisseriafor example N. gonorrhoeae; Escherichia coli for example E. coliO157:H7; Shigella for example S. enteritis and S. flexneri;Campylobacter for example C. jejuni; Yersinia for example Y.pseudotuberculosis and Y. pestis; Listeria for example L. monocytogenes;Vibrio for example V. cholerae; and the like. In a related embodiment,the infectious agent is at least one virus selected from: humanimmunodeficiency virus (HIV); influenza virus for example influenza A orB, for example A/H1N1; polio; herpes for example Herpes simplex virus-1and Herpes simplex virus-2; smallpox; measles; mumps; rubella;rotavirus; chicken pox; rabies; West Nile virus; Ebola for example Ebolahemorrhagic fever; eastern equine encephalitis; norovirus; hepatitis forexample Hepatitis A, Hepatitis B, and Hepatitis C; and the like. In arelated embodiment, the infectious agent is at least one fungus selectedfrom: Cryptococcus for example C. Gattii and C. neoformans v.neoformans; Candida for example C. albicans; Aspergillus for example A.flavus and A. fumigatus; and the like. In a related embodiment, theinfectious agent is at least one protozoan selected from: Entamoeba forexample E. histolytica; Giardia for example G. lamblia; Cryptosporidiumfor example C. parvum; Naegleria for example N. fowleri and N. gruberi;and the like.

In a related embodiment of the method, the antigen is a rotavirusantigen, for example of bovine, human, or murine origin, or the antigenis a bacterial toxin antigen, for example, a Clostridium tetani tetanustoxin antigen. In related embodiments, the rotavirus antigen is a viralvirion protein, for example the viral virion protein is selected from:VP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof.

In a related embodiment, the allergen includes a macromolecule orportion thereof associated with an increased immunoglobulin level orallergic response in the subject, for example the allergen includes anenvironmental allergen, animal or plant allergen, or food allergen, forexample the allergen is associated with pollen for example ragweed, dustmite proteases for example as found in dust mite excretion, fungus forexample mold, pet dander or saliva, shellfish, seafood, a legume such aspeanuts, and the like.

In a related embodiment of the method, the subject is a vertebrateanimal. For example the vertebrate animal is selected from: anagricultural animal, a high value zoo animal, a research animal, ahuman, and a wild animal in a dense human environment. For example thevertebrate animal is a cow, a dog, or a pig.

In a related embodiment, contacting the mucosal tissue of the subjectfurther involves administering the composition by a route selected from:intravenous, intramuscular, intraperitoneal, intradermal,intrapulmonary, intravaginal, rectal, oral, buccal, sublingual,intranasal, intraocular, and subcutaneous.

In a related embodiment, contacting the mucosal tissue of the subjectinvolves applying to the mucosal tissue at least one of: an aerosol, amist, a nose drop, an eye drop, a mouth drop, a capsule, a tablet, apill, a powder, a granule, a fluid, a suspension, an emulsion, a gel, apatch, and a lozenge.

In a related embodiment, the method further involves after providing thevegetative host bacterial cell and contacting the mucosal tissue of thesubject, immunizing the subject with the host bacterial cell, forexample the host bacterial cell includes a Bacillus cell. For example,the Bacillus is Bacillus subtilis.

In a related embodiment of the method, contacting the mucosal tissue ofthe subject further includes contacting the mucosal tissue with anadjuvant. In a related embodiment of the method, the adjuvant isselected from: cholera toxin, a non-toxic variant of Escherichia colilabile toxin, and a portion or a derivative thereof.

In a related embodiment, the method further includes prior to contactingthe mucosal tissue of the subject, lyophilizing the composition. Forexample, the composition is lyophilized and distributed under vacuum ina tube or vial.

In a related embodiment, the method further includes observingresistance of the composition to at least one condition selected from:heat, drying, freezing, deleterious chemicals, and radiation.Alternatively, the method includes observing resistance of thecomposition to accumulation of moisture. In a related embodiment of themethod, the resistance to heat includes observing resistance at 60° C.or 45° C. for hours, days, months, for example at least six months, oryears, for example at least one year or at least two years. In a relatedembodiment of the method, observing resistance includes observing a heattreated composition maintaining ability to confer on the subject fullprotective immunity or at least partial protective immunity, such thatthe partial protective immunity includes a percentage of the fullprotective immunity, such that the percentage includes at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

In a related embodiment, the method further includes: measuring anantibody titer in serum of the subject administered the composition,such that an increase in antibody for the antigen in comparison to acontrol serum not administered the composition is an indication ofefficacy of the immunogenicity of the composition. For example, theantibody is anti-TetC IgG or IgA.

In a related embodiment, the method further includes: measuring anamount of antigen shedding in the subject having been afflicted by theinfectious agent, such that a decrease in fecal antigen as compared tothat in a control also afflicted by the infectious agent and notcontacted with the composition is a measure of efficacy of theimmunogenicity of the composition.

In a related embodiment of the method, the isolated nucleotide sequenceencodes an antigen of a Clostridium tetani tetanus toxin antigen, suchthat the nucleotide sequence is operably linked to a promoter forcytoplasmic vegetative expression of the Clostridium tetani tetanustoxin antigen or for expression of the Clostridium tetani tetanus toxinantigen as a fusion to a spore coat protein, such that at least one ofvegetative cells and spores are associated with the Clostridium tetanitetanus toxin antigen.

An embodiment of the invention provides a thermally-stable vaccinecomposition for immunizing a subject with an antigen from an infectiousagent, a tumor, or an allergen, the composition including at least oneof vegetative cells and spores from a Bacillus cell, such that the cellsinclude an isolated nucleotide sequence encoding the antigen, thenucleotide sequence being genetically engineered and integrated into thehost bacterial chromosome or carried on a plasmid and provided withappropriate transcriptional and translational regulatory sequences, sothat the cells express the antigen cytoplasmically during vegetativegrowth, or the cells express the antigen during sporulation as a geneticfusion to a spore coat protein so that upon sporulation by the cells theantigen is associated with the vegetative cells, the spores, or both,and such that the composition is effective when applied to a mucosaltissue of the subject to immunize the subject from the infectious agent,tumor, or the allergen.

In a related embodiment, the Bacillus is Bacillus subtilis. In a relatedembodiment, the composition further includes an adjuvant. For example,the adjuvant is selected from at least one of: cholera toxin, anon-toxic variant of Escherichia coli labile toxin, and a portion or aderivative thereof.

In a related embodiment of the composition, the isolated nucleotidesequence encoding the antigen which is from a strain that is mammalian,for example bovine, human or murine.

In a related embodiment, the composition is treated to removesubstantially all water by at least one of technique, for example,centrifugation, vacuum, lyophilization, spray drying, and the like. Forexample, the composition is dried at 37° C. overnight.

In a related embodiment of the composition, the infectious agent isselected from the group of: a bacterium, a fungus, a virus, a protozoan,or a protein product thereof. In a related embodiment of thecomposition, the infectious agent is at least one bacterium selectedfrom: Bacillus for example B. anthracis; Clostridium for example C.tetani, C. difficile, and C. perfringens; Corynebacterium for example C.diphtheriae; Bordetella for example B. pertussis; Mycobacterium forexample M. tuberculosis; Salmonella for example S. enterica;Staphylococcus for example S. aureus and S. epidermis; Streptococcus forexample S. pneumoniae and S. mutans; Treponema for example T. pallidum;Plasmodium for example P. falciparum, P. vivax, P. malariae, and P.ovale; Pseudomonas for example P. aeruginosa; Neisseria for example N.gonorrhoeae; Escherichia coli for example E. coli O157:H7; Shigella forexample S. enteritis and S. flexneri; Campylobacter for example C.jejuni; Yersinia for example Y. pseudotuberculosis and Y. pestis;Listeria for example L. monocytogenes; Vibrio for example V. cholerae;and the like.

In a related embodiment of the composition, the infectious agent is atleast one virus selected from the group of: human immunodeficiency virus(HIV); influenza virus for example influenza A or B, for example A/H1N1,polio; herpes for example Herpes simplex virus-1 and Herpes simplexvirus-2; smallpox; measles; mumps; rubella; rotavirus; chicken pox;rabies; West Nile virus; Ebola for example Ebola hemorrhagic fever;eastern equine encephalitis; norovirus; hepatitis for example HepatitisA, Hepatitis B, and Hepatitis C; and the like.

In a related embodiment of the composition, the infectious agent is atleast one fungus selected from: Cryptococcus for example C. Gattii andC. neoformans v. neoformans; Candida for example C. albicans;Aspergillus for example A. flavus and A. fumigatus; and the like.

In a related embodiment of the composition, the infectious agent is atleast one protozoan selected from the group of: Entamoeba for example E.histolytica; Giardia for example G. lamblia; Cryptosporidium for exampleC. parvum; Naegleria for example N. fowleri and N. gruberi; and thelike.

In a related embodiment of the composition, the allergen includes amacromolecule or portion thereof associated with an increasedimmunoglobulin level or allergic response in the subject, for examplethe allergen includes an environmental allergen, animal or plantallergen, or food allergen, for example the allergen is associated withpollen for example ragweed, dust mite proteases for example as found indust mite excretion, fungus for example mold, pet dander or saliva,shellfish, seafood, a legume such as peanuts, and the like.

In a related embodiment of the composition, the tumor is associated withat least one cancer. For example, the cancer is a disease of: whiteblood cells for example a lymphocyte, skin, eye, mouth, brain,esophagus, breast, lung, liver, pancreas, stomach, colon, kidney,bladder, ovary, cervix, and vagina. For example the antigen of the tumoris at least one selected from the group of: alphafetoprotein (AFP),human epidermal growth factor receptor 2 (HER2), nestin,carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125), humanchorionic gonadotropin (HCG), epithelial tumor antigen (ETA),melanoma-associated antigen (MAGE), tyrosinase related protein 1(TRP-1), G melanoma antigen (GAGE), B melanoma antigen (BAGE),cyclin-dependent kinase 4 (CDK4), and beta-catenin.

In a related embodiment, the composition is formulated to beadministered by at least one route selected from: intravenous,intramuscular, intraperitoneal, intradermal, intrapulmonary,intravaginal, rectal, oral, buccal, sublingual, intranasal, intraocular,and subcutaneous.

An embodiment of the invention provides a vaccination kit that includesa unit dose of the composition according to any of embodiments herein, acontainer, and instructions for use.

In a related embodiment of the kit, the instructions include storage ata room temperature from about 4° C. to about 45° C. and the like. In arelated embodiment, the composition is heat stable at 4° C. to about 45°C. for a period of time for example days, weeks, months, or years.

In a related embodiment, the kit further includes an applicator for thecomposition, for example, a spray bottle, a nasal sprayer, a fluiddropper, an oral inhaler, a nasal inhaler, or a syringe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph showing serum antibody titer to rotavirus VP6observed following intranasal immunization of mice with B. subtiliscontrol spores (squares) or B. subtilis spores associated with bovine(triangles) or murine (circles) VP6 antigen as a function of time (daysafter the first immunization). B. subtilis spores were administered withcholera toxin (open symbols) or without cholera toxin (closed symbols)as an adjuvant. The data show that animals administered VP6 antigenproduced antibody, and that antibody titer was improved by use of theadjuvant.

FIG. 2 is a line graph showing amount of rotavirus in feces as afunction of time (days) after rotavirus challenge of mice previouslyimmunized with each of: B. subtilis control spores (squares) and B.subtilis spores associated with either VP6 antigen from rotavirusstrains of bovine origin (triangles) or with rotavirus of murine origin(circles). The B. subtilis spores were administered with cholera toxin(ct; open symbols) or without cholera toxin (closed symbols). Thedisease model was epizootic diarrhea of infant mice (EDIM). The datashow that fecal viral content was reduced in EDIM mice previouslyinoculated with murine VP6 associated spores or with bovine VP6associated spores, compared to control spores, and that adjuvant furtherdecreased the fecal viral content.

FIG. 3 is a line graph showing serum antibody titer specific forrotavirus VP6 observed after mice were immunized intranasally with B.subtilis control spores (squares) or B. subtilis spores associated withbovine derived VP6 (circles, diamonds) or murine derived VP6 (triangles)as a function of time (days after first immunization). B. subtilisspores were administered with an adjuvant prepared from non-toxicEscherichia coli LT (R192G having a mutation of arginine to glycine atresidue 192) at 5 μg/dose or 10 μg/dose. The data show that an amount of5 μg/dose or 10 μg/dose of LT (192G) was effective as an adjuvant. Noserum antibody titer was observed in animals administered the controlspores, even in the presence of the adjuvant.

FIG. 4 is a line graph showing amount of rotavirus in feces as afunction of time (days) after rotavirus challenge of mice immunized withB. subtilis control spores (squares) or B. subtilis spores associatedwith VP6 antigen of rotavirus strains of bovine origin (circles,diamonds) or murine origin (triangles). Spores were administered with anadjuvant prepared from non-toxic Escherichia coli LT (R192G) at 5μg/dose or 10 μg/dose. The data show that the mice immunized with B.subtilis spores that were associated with bovine or murine VP6 recoveredmore quickly from the rotavirus infection than control mice, and thatfeces produced by the VP6-immunized mice contained far fewer virusparticles than feces from the mice administered control spores.

FIG. 5 is a listing of the nucleotide sequence (SEQ ID No: 1) of themodified Pspac promoter for expression of antigens constitutively at ahigh level.

FIG. 6 is a set of photographs showing expression of tetanus toxinC-terminal fragment (TTFC synonymous with TetC) in recombinant B.subtilis. Colonies grown of solid medium were labeled with rabbitanti-tetanus toxin (TT) antibody followed by anti-rabbit IgG-FITCconjugate of TTFC-expressing strain BB2646 but not control BB2643strain, and expression by colonies was observed by presence offluorescein stain. FIG. 6 shows Coomassie blue stained 4-12% SDS-PAGE(left panel), and TTFC-specific Western blot (right panel) profiles offractionated cell extracts from BB2643 (control) and BB2646(TTFC-expressing). Arrows indicate TTFC at the predicted molecularweight of 50 KDa.

FIG. 7 is a bar graph showing serum anti-TTFC antibody titers after oralimmunization of BALB/c mice with each of strains BB2646 carrying tetanustoxin C-terminal fragment (TetC synonymous with TTFC), and strainBB2643, a negative control.

FIG. 8 is a bar graph showing serum anti-TTFC antibody titers afterintranasal immunization of BALB/c mice with B. subtilis vegetative cellsexpressing TetC cytoplasmically or after intra-muscular (i.m.)immunization with a conventional DTaP vaccine (positive control). Forimmunization with B. subtilis, mice were inoculated intranasally with1×10⁸ cells in a volume of 20 μl on days 0, 14, and 28 or on days 0, 2,14, 16, 28, and 30. For DTaP vaccination, mice were injected i.m. with50 μl of DTaP vaccine as provided by the manufacturer. Arrows indicatemice that died after challenge.

FIG. 9 is a set of line graphs and a bar graph showing dose response ofimmune response generated by spore preparations of strain BB2646 andprotection against lethal tetanus toxin challenge in BALB/c mice. Eachimmunized mouse was tested for immune response by intraperitonealinjection with an amount of tetanus toxin equivalent to twice the 100%lethal dose (LD₁₀₀) of tetanus toxin and was examined for symptoms atthe time indicated on the abcissa. It was observed that more than 10⁹spores were required for effective immunization. FIG. 9 panel A is aline graph showing average serum anti-TetC antibody titers in animalsadministered with 10⁹ spores in control (closed squares),TTFC-associated spores at concentrations of: 10⁷ (open squares), 10⁸(closed triangles) and 10⁹ (open triangles). The arrows indicate thetime points of immunization. FIG. 9 panel B is a bar graph showingindividual serum anti-TetC antibody titers in four groups of animalsimmunized with control and TTFC-associated spores at threeconcentrations 10⁷, 10⁸ and 10⁹. FIG. 9 panel C is a line graph showingmouse survival in four groups of animals challenged with 10⁹ spores incontrol (closed squares), TTFC-associated spores at concentrations of:10⁷ (open squares), 10⁸ (closed triangles) and 10⁹ (open triangles). Itwas observed that mouse survival rate was 100% after immunization with10⁹ TTFC associated spores.

FIG. 10 is a line graph and a bar graph showing effect of incubation at37° C. on immunogenicity of BB2646 spores. It was observed thatimmunogenicity was stable at 4° C. after lyophilization, but not inliquid suspension after storing for 5 weeks at 37° C. FIG. 10 panel A isa line graph showing anti-TetC antibody titers in animals immunized withcontrol spores (closed squares), TTFC-associated spores: not treated(open squares), treated with 37° C. for 5 weeks (closed triangles) andlyophilized (open triangles). The arrows indicate the dates ofimmunization. FIG. 10 panel B is a bar graph showing individual serumanti-TetC antibody titers after immunization of four groups of animalswith spores stored at various conditions compared to animals immunizedwith control spores.

FIG. 11 is a set of graphs showing role of spore germination inimmunogenicity of BB2646 spores. FIG. 11 panel A is a line graph showingaverage serum anti-TetC antibody titers in animals immunized withcontrol spores (closed squares), TTFC-associated germinating spores(open triangles) and TTFC-associated germination deficient spores (opencircles). FIG. 11 panel B is a bar graph showing individual serumanti-TetC antibody titers with germinating and germination deficientspores compared to control spores. FIG. 11 panel C is a line graphshowing survival rate in challenged animals immunized with controlspores (closed squares), TTFC-associated germinating spores (opentriangles) and TTFC-associated germination deficient spores (opencircles). It was observed that spore germination was not required forimmunization.

FIG. 12 is a line graph and a bar graph showing antibody endpoint titers(panel A) and survival rate (panel B) of mice following intranasalimmunization with vegetative cells of strain BB2646 expressing TetC incytoplasm, and controls. Survival in mice receiving 10⁸ (open squares)spores and mice receiving intramuscular (IM) injection ofDTaP-associated spores was 100%, compared to lower survival levels inmice receiving 10⁷ (open triangles) and control (closed diamonds)animals.

FIG. 13 is a bar graph and a line graph showing a relationship betweenimmunogenicity and germination of BB2646 spores and outgrowth ofvegetative cells. FIG. 13 panel A shows individual serum anti-TetCantibody titers in mice after three rounds of inoculation withTTFC-associated spores before or 1-3 hours after suspension in growthmedium. FIG. 13 panel B shows mouse survival levels followingimmunization with 10⁹ untreated dormant (bright) (closed triangles), 10⁹dormant (bright) spores heated to 80° C. for 10 min before inoculation(open triangles), 10⁹ germinated (dark) spores after incubation for 1 hin growth medium (closed circle), 10⁹ germinated (dark) spores incubatedfor three hours in growth medium (closed squares) and 10⁸ germinated(dark) spores incubated for three hours in growth medium (open squares).Highest titers and greatest survival was observed in mice inoculatedwith 10⁹ spores incubated for three hours in growth medium. It wasobserved that the population of cells in this population hadsubstantially converted to vegetative cells.

FIG. 14 panel A is a line graph showing serum anti-TetC antibody titersafter intranasal immunization of BALB/c mice with dried, heated B.subtilis vegetative cells expressing TTFC cytoplasmically (open circles)or control (closed squares). The dried vegetative cells were treated at60° C. for 1 hr and resuspended in sterile H₂O before immunization. Micewere inoculated intranasally in a volume of 20 μl per dose on days 0,14, and 28. Serum titer in mice immunized with TTFC was five orders ofmagnitude greater than in control mice.

FIG. 14 panel B is a line graph showing protection against lethaltetanus toxin challenge in BALB/c mice after intranasal immunizationwith dried, heated B. subtilis vegetative cells expressing TTFCcytoplasmically (open circles) or control (closed squares). Each mousewas injected intraperitoneally with a dose of two LD₁₀₀ amount oftetanus toxin and was examined for symptoms during the time periodindicated. Data show survival of immunized mice

FIG. 15 is a set of line graphs showing serum anti-TetC antibody titersand survival rates in mice after intranasal immunization of BALB/c micewith dried, heated B. subtilis spores displaying TTFC on the sporesurface, compared to control spores. FIG. 15 panel A is a line graphshowing serum anti-TetC antibody titers in animals immunized withTTFC-displaying spores that were either dried and heated to 60° C. for60 min (open diamonds) or untreated (open circles) in comparison withcontrol spores that were dried and heated (open squares). FIG. 15 panelB is a line graph showing survival rate in animals inoculated withTTFC-displaying spores that were either dried and heated 60° C. for 60min (open diamonds) or untreated (open circles) in comparison to controlspores that were dried and heated (open squares). It was observed thatheating spores in the dry state did not diminish the immune response inmice.

FIG. 16 is a line graph showing development of antibody response ingroups of mice inoculated with vegetative cells of strain BB3059, whichcontains three copies of the Pspac-tetC construct: freshly grown,unheated BB3059 vegetative cells (open squares), 4×10⁸ lyophilizedBB3059 cells incubated at 45° C. for 30 days (open circles), 4×10⁷lyophilized BB3059 cells at 45° C. for 30 days (gray filled circles),4×10⁶ lyophilized BB3059 cells incubated at 45° C. (black filledcircles) compared to unheated, freshly grown vegetative cells of thecontrol strain BB2643 (closed squares) and cells immunized IP with DTaPvaccine (open diamonds). Long-term heat stability of lyophilized cellsof strain BB3059 incubated at 45° C. for one month was observed.

FIG. 17 is a set of line graphs showing antibody development in micetreated with spores incubated at 45° C. for one month (panel B) comparedto control (panel A), demonstrating long-term heat stability of strainBB3184, which contains three copies of the cotC-tetC construct. FIG. 17panel A shows the immune response in animals immunized with CotC-TetCspores in H₂0 (open squares), 10⁹ lyophilized spores (open circles), 10⁸lyophilized spores (gray filled circles), 10⁷ lyophilized sporescompared to spores of the control strain BB2643 (closed squares). FIG.17 panel B shows immune response in animals treated with CotC-TetCspores lyophilized and stored at 4° C. (open squares), 10⁹ sporeslyophilized and incubated at 45° C. for one month (open circles), 10⁸spores lyophilized and incubated at 45° C. for one month (gray filledcircles) in comparison with spores of the control strain BB2643 storedin H₂O at 4° C. (closed squares).

FIG. 18 is a scatter graph showing IgG1:G2a ratio observed in serumafter intranasal immunization with B. subtilis BB3059 vegetative cells,and control i.m. injection of DTaP.

FIG. 19 is a bar graph showing IgA levels in serum and feces of micefollowing intranasal immunization with B. subtilis spores associatedwith bovine VP6 antigen from construct BB2666 in the presence of mLTadjuvant. The data show that immunized animals produced higher levels ofIgA in feces than in serum.

FIG. 20 is a bar graph showing IgA levels following intranasalimmunization of mice with B. subtilis spores (light gray bars) or B.subtilis vegetative cells (dark gray bars) associated with bovine VP6antigen from construct BB2666. Spores and vegetative cells wereadministered with mLT adjuvant. The data show that animals administeredspores associated VP6 produced higher levels of IgA than animalsadministered with vegetative cells.

FIG. 21 is a set of drawings showing a schematic growth curve of B.subtilis vegetative cells and initiation of sporulation after onset ofgrowing phase. FIG. 21 panel A shows antigen display on surface of thespore. FIG. 21 panel B shows antigen displayed on the vegetative cellsurface. FIG. 21 panel C shows antigen displayed in the vegetativecytoplasm.

FIG. 22 is a set of drawings showing construction of genetic fusions forantigen display. FIG. 22 panel A shows organization of a recombinantfusion of an exemplary gene CotC with TetC coding gene. FIG. 22 panel Bshows organization of an exemplary gene for expressing antigen duringvegetative growth.

FIG. 23 is a set of line graphs showing serum titer (ordinate, logscale) response after spore immunization with spores carrying a tetanussequence fusion (CotC-TetC).

FIG. 23 panel A shows higher serum levels in mice immunized with 10⁹spores of lyophilized strain BB3184 CotC-TetC fusion heated to 45° C.for 30 days (open circles) or 10⁹ spores of lyophilized strain BB3184stored at 4° C. for 30 days (open squares) or with 10⁸ spores oflyophilized strain BB3184 TetC heated to 45° C. (closed diamonds) for 30days compared to the control vegetative cells (closed squares). Sporesof strain BB3184 heated or stored for 30 days induced antibodyproduction.

FIG. 23 panel B shows that spores of strain BB3184 heated or stored for90 days were immunized for anti-tetanus antibody production.

FIG. 24 is a line graph showing log of titer of antibody titer in micethat were immunized intranasally with: 10⁹ spores of strain BB3184stored at 4° C. for 12 months (open squares), 10⁹ spores of strainBB3184 heated to 45° C. for 12 months (open circles), 10⁸ spores ofstrain BB3184 heated to 45° C. for 12 months (closed diamonds), orcontrol spores (closed squares). Data show that spores heated to 45° C.for 12 months induced antibody production as well as or better thanspores stored at 4° C.

FIG. 25 is a line graph showing survival of number of mice intranasallyimmunized with lyophilized spores and challenged with then a lethal doseof tetanus toxin. The mice were immunized intranasally with: 10⁹ sporesof strain BB3184 cotC-TetC stored at 4° C. for 12 months (open squares),10⁹ spores of strain BB3184 heated to 45° C. for 12 months (opencircles), 10⁸ spores of strain BB3184 heated to 45° C. for 12 months(closed diamonds), or control spores (closed squares). Data show 100%survival of subject immunized with 10⁹ spores of strain BB3184 heated to45° C. for 12 months or control 10⁹ spores of strain BB3184 heated to 4°C. for 12 months. Animals immunized with 10⁸ spores of strain BB3184heated to 45° C. for 12 months resulted in 60% survival day three afterchallenge and 40% survival day seven after challenge. Animalsadministered control spores not carrying TetC did not survive beyond day3 after challenge. The lyophilized vaccine heated to 45° C. for 12months provided as much protective immunogenicity as that stored at 4°C.

FIG. 26 is a photograph of a Western blot showing expression of TetC inrecombinant B. subtilis vegetative cell strains. Data show that TetCantigen was produced in large quantities in the vegetative cells (theantigen represents about three percent of the total soluble protein).

FIG. 27 is a set of photographs of a Western blot and a Coomassieblue-stained gel showing expression of TetC antigen in each of arecombinant B. subtilis vegetative cell strain producing toxin antigenunder regulation of the IPTG-inducible Pspac, and in B. subtilis sporesas fusion cotC-TetC. The vegetative cell and spore vaccines wereincubated for at least five hours at 37° C., and samples taken duringthis time course were analyzed by Western blot and Coomassie.

FIG. 28 is a line graph showing log serum titer response afterlyophilized vegetative cell immunization in groups of mice. The micewere immunized intranasally with: 4×10⁸ lyophilized cells of strainBB3059 stored at 4° C. for 90 days (open squares), 4×10⁸ lyophilizedcells of strain BB3059 heated at 45° C. for 90 days (open circles),4×10⁷ cells of strain BB3059 heated at 45° C. for 90 days (closedcircles), or control lyophilized vegetative cells not expressing TetC(closed squares). Data show that lyophilized vegetative cells of strainBB3059 stored for three months at 45° C. or 4° C. elicited the highestserum titer compared to the control vegetative cells. Thus, lyophilizedvegetative cells expressing TetC stored at 4° C. for 90 days or heatedto 45° C. for 90 days were comparably stable vaccines.

FIG. 29 is a set of line graphs of log serum titer response in miceimmunized with lyophilized vegetative cells expressing TetC. The micewere immunized intranasally with: 4×10⁸ cells of strain BB3059 stored at4° C. for twelve months (open squares), 4×10⁸ cells of strain BB3059heated to 45° C. for twelve months (open circles), 4×10⁷ cells of strainBB3059 heated at 45° C. for twelve months (open diamonds), or controllyophilized vegetative cells not expressing TetC (closed squares). Theserum was assayed for titer of anti-tetanus toxin.

FIG. 29 panel A shows that 4×10⁸ cells of lyophilized vegetative cellsof strain BB3059 stored at 4° C. and heated at 45° C. for twelve monthshad a higher serum titer than that induced with 4×10⁷ lyophilizedvegetative cells of strain BB3059 heated to 45° C. for twelve months andthe control lyophilized vegetative cells. Thus the lyophilizedvegetative cells of strain BB3059 were stable as vaccines when stored at4° C. or heated at 45° C. for 12 months.

FIG. 29 panel B shows 100% survival rates for subjects immunized withTetC expressing lyophilized vegetative cells of strain BB3059, and zerosurvival rate days for animals immunized with control cells.

FIG. 30 is a set of line graphs showing serum titer response for miceinoculated intranasally (IN) or sublingually (SL) with lyophilized B.subtilis vegetative cells of strain BB3059 expressing TetC that werelyophilized and heated at 45° C. for 17 months. Mice were immunized:intranasally with lyophilized vegetative cells of strain BB3059 heatedat 45° C. for 17 months (open squares), sublingually with controllyophilized vegetative cells heated at 45° C. for 17 months (opentriangles), or intranasally with control lyophilized vegetative cells ofstrain BB3059 (closed circles).

FIG. 30 panel A shows that the average serum titer data of miceimmunized intranasally with lyophilized vegetative cells of strainBB3059 produced higher serum titer than mice immunized sublingually withlyophilized vegetative cells of strain BB3059 or control lyophilizedvegetative cells of strain BB2643. Thus, lyophilized vegetative cells ofstrain BB3059 expressing TetC were heat stable for 17 months, andcapable of eliciting anti-TetC antibodies following IN or SLadministration.

FIG. 30 panel B shows individual animal serum titer data including thatthe animals immunized intranasally (IN) or sublingually (SL) withlyophilized B. subtilis vegetative cells of strain BB3059 expressingTetC. Higher serum titer values were obtained in individual mice thatwere intranasally immunized compared to individual mice sublinguallyimmunized.

FIG. 31 is a chart showing an intranasal vaccination chart and schedulefor normal mice and SCID immune deficient/human immune system mice.

FIG. 32 is a set of line graphs showing body weight (grains, g;ordinate) of inoculated mice as a function of time (days, abscissa)after inoculation with Bacillus lyophilized cells of strain 49NA2 (opentriangles) or with spores of strain 49NB3 (open squares).

FIG. 32 panel A shows that body weight of normal Balb/cByJ miceincreased as a function of time (32 days).

FIG. 32 panel B shows that body weight of SCID CBySmn.CB17-Prkdcsid/Jmice increased as a function of time (32 days).

FIG. 33 is a set of line graphs showing serum titer response and numberof surviving mice (survival). The animals were sublingually (SL)immunized with lyophilized B. subtilis vegetative cells strain BB3059with or without mutated E. coli heat labile enterotoxin (mLT) adjuvant,then challenged with a lethal dose of tetanus toxin.

FIG. 33 panel A is a line graph showing average serum anti-TetC antibodytiters for immunized animals. The animals were immunized: sublinguallywith: lyophilized B. subtilis vegetative cells strain BB3059 TetC(closed circles), lyophilized B. subtilis vegetative cells BB3059 TetCand mLT adjuvant (closed squares); control lyophilized B. subtilisvegetative cells (open triangles), or control lyophilized B. subtilisvegetative cells and mLT adjuvant (open diamonds). Data show thatanimals sublingually immunized with lyophilized B. subtilis vegetativecells BB3059 expressing TetC (70B BB3059 SL Veg cells) showed greaterserum antibody titer than animals administered control lyophilizedvegetative cells, regardless of presence of mLT adjuvant

FIG. 33 panel B is a line graph showing mouse survival rate followingchallenge with lethal tetanus toxin of mice immunized sublingually with:control lyophilized vegetative cells (open triangles), lyophilized B.subtilis vegetative cells BB3059 expressing TetC (closed circles), orlyophilized B. subtilis vegetative cells BB3059 expressing TetC, and mLTadjuvant (closed squares). Data show 100% survival rates through day 10for animals immunized with TetC expressing lyophilized vegetative cellsof strain BB3059, and 40% survival rate through day ten after challengeof animals immunized with TetC expressing lyophilized vegetative cellsof strain BB3059 and mLT adjuvant. Control lyophilized vegetative cellsprovided no immunity.

FIG. 34 is a set of line graphs showing serum titer response andsurvival rate to lethal tetanus toxin challenge following sublingualimmunization with lyophilized B. subtilis vegetative cells compared topurified recombinant TetC.

FIG. 34 panel A shows average serum anti-TetC antibody titers in animalsimmunized: sublingually with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC (open squares); intranasally with lyophilized B.subtilis vegetative cells BB3059 expressing TetC (open diamonds);sublingually with purified recombinant TetC (open triangles); orsublingually with control lyophilized B. subtilis vegetative cells(closed circles). Animals immunized intranasally with lyophilized B.subtilis vegetative cells BB3059 expressing TetC, animals immunizedsublingually with either lyophilized B. subtilis vegetative cells BB3059expressing TetC, and animals immunized with recombinant TetC allproduced comparably high levels of anti-TetC antibody.

FIG. 34 panel B is a line graph showing mouse survival rate (%;ordinate) as a function of time (days; abscissa) for mice challengedwith lethal tetanus toxin and previously immunized: sublingually withlyophilized B. subtilis vegetative cells BB3059 expressing TetC (opensquares); intranasally with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC (open diamonds); sublingually with purifiedrecombinant TetC (open triangles); or sublingually with controllyophilized B. subtilis vegetative cells (closed circles). Data show100% survival of animals immunized either intranasally or sublinguallywith lyophilized B. subtilis vegetative cells BB3059 expressing TetC,compared to a 80% survival rate for animals immunized sublingually withrecombinant TetC, and no survival for animals immunized with controllyophilized vegetative cells.

FIG. 35 is a set of bar graphs showing serum IgG1 levels (open bars;abscissa) and IgG2a levels (closed bars; abscissa) in mice followingsublingual immunization with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC (TetC veg Cells, SL); intranasal immunizationwith lyophilized B. subtilis BB3059 expressing TetC (TetC veg Cells,IN); sublingual immunization with purified recombinant TetC (rTTC, SL);or intramuscular immunization with a commercial vaccine for diphtheria,tetanus and pertussis (DTAP, IM; Tripedia®, Sanofi Pasteur Inc.,Swiftwater, Pa., USA). Each animal showed greater IgG1 levels comparedto IgG2a levels. Sublingual or intranasal immunizations of animals withlyophilized B. subtilis BB3059 TetC resulted in more similarTetC-specific IgG1 and IgG2a antibody production compared to therecombinant TetC or commercially available DTAP vaccine.

FIG. 36 is a set of bar graphs showing mouse serum concentrations ofcytokines (ng/mL; ordinate) two weeks following a third immunization(abscissa). Mice were immunized sublingually with lyophilized B.subtilis vegetative cells BB3059 expressing TetC (TetC veg cells, SL);intranasally with lyophilized B. subtilis vegetative cells BB3059expressing TetC (TetC veg cells, IN); sublingually with purifiedrecombinant TetC (rTTC); or intramuscularly with commercial DTAP vaccine(DTAP).

FIG. 36 panel A shows substantial interleukin-2 (IL-2) in serum ofanimals sublingually or intranasally immunized with lyophilized B.subtilis vegetative cells BB3059, comparable to animals immunizedsublingually with either purified recombinant TetC and or DTAP vaccine.

FIG. 36 panel B shows greater interferon-gamma (IFNγ) serumconcentrations in serum of animals sublingually immunized withlyophilized B. subtilis vegetative cells BB3059 expressing TetC comparedto animals immunized intranasally with lyophilized B. subtilisvegetative cells BB3059 expressing TetC, sublingually with purifiedrecombinant TetC, or intramuscularly with DTAP vaccine.

FIG. 36 panel C shows greater interleukin-4 (IL-4) serum concentrationsin serum of animals intranasally immunized with lyophilized B. subtilisvegetative cells BB3059 expressing TetC than animals immunizedintramuscularly with DTAP vaccine, or sublingually immunized with eitherlyophilized B. subtilis vegetative cells BB3059 expressing TetC orpurified recombinant TetC.

FIG. 36 panel D shows comparable levels of serum interleukin-10 (IL-10)in animals immunized sublingually with purified recombinant TetC, andanimals sublingually, intranasally immunized with lyophilized B.subtilis vegetative cells BB3059 and animals immunized sublingually withDTAP vaccine.

FIG. 37 is a set of bar graphs showing amount of fecal immunoglobulin inmice immunized sublingually with control lyophilized vegetative cells(Ctl veg cells SL), sublingually with lyophilized B. subtilis vegetativecells BB3059 expressing TetC (TetC veg cells, SL), intranasally withlyophilized B. subtilis vegetative cells BB3059 expressing TetC (TetCveg cells, IN), or intramuscularly with commercial DTAP vaccine (DTAP,IM).

FIG. 37 panel A shows high IgG fecal content in mice inoculatedsublingually with lyophilized B. subtilis vegetative cells of strainBB3059 expressing TetC, mice inoculated intramuscularly with commercialDTAP vaccine or mice inoculated sublingually with lyophilized B.subtilis vegetative cells of strain BB3059 expressing TetC compared toIgG fecal content in mice inoculated sublingually with controllyophilized B. subtilis vegetative cells.

FIG. 37 panel B shows high IgA fecal content in mice inoculatedintranasally with lyophilized B. subtilis vegetative cells BB3059expressing TetC, mice inoculated sublingually with lyophilized B.subtilis vegetative cells BB3059 expressing TetC compared to IgA fecalcontent in mice inoculated with control lyophilized B. subtilisvegetative cells and commercial DTAP vaccine.

FIG. 38 is a set of bar graphs showing amount of TetC specific IgAdetected by ELISA in vaginal and saliva samples of mice immunized:sublingually with lyophilized B. subtilis vegetative cells BB3059expressing TetC (open), intranasally with lyophilized B. subtilisvegetative cells of strain BB3059 expressing TetC (diagonal),sublingually with purified recombinant TetC (checkered), orintramuscularly with commercial DTAP vaccine (closed). Data show higheramounts of TetC specific IgA detected in the vaginal samples compared tothe in saliva samples, and highest amounts in animals inoculatedsublingually with lyophilized B. subtilis vegetative cells of strainBB3059 expressing TetC, compared to intranasally, and in mice inoculatedsublingually with purified recombinant TetC, or intramuscularly withcommercial DTAP vaccine.

FIG. 39 is a set of representative photomicrographs showing MHC class IIdeposition of murine spleen tissue (panels A and B respectively), andmurine intestinal tract tissue (panels C and D) in animals sacrificed 24hours after immunization. The animals were sublingually immunized witheither lyophilized B. subtilis vegetative cells BB3059 expressing TetC(panels A and C) or with control lyophilized B. subtilis vegetativecells (panels B and D).

FIG. 39 panels A and C show MHC class II deposition on murine spleentissue and murine intestinal tract tissue, respectively, from micesublingually immunized with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC. Extensive MAC immunostaining with variouspatterns of staining of spleen cells was observed.

FIG. 39 panels B and D show MHC class II deposition on murine spleentissue and murine intestinal tract tissue, respectively, from micesublingually immunized with control lyophilized B. subtilis vegetativecells. Little or no MHC class II immunostaining of cells was observed onthe murine intestinal tract tissue.

FIG. 40 is a bar graph showing anti-LT specific IgA and IgGimmunoglobulin levels (absorbance at 450 nm; ordinate) in serum, fecaland saliva samples of piglets (abscissa) 14 days after a fourthsublingual immunization with lyophilized B. subtilis vegetative cellswith (open bars) or without mLT adjuvant (closed bars). Animalsadministered mLT adjuvant received 25 μg of the mLT adjuvant perinoculation. The data show that in each of the serum, fecal, and salivasamples animals administered lyophilized B. subtilis vegetative cellswith mLT adjuvant produced higher levels of IgG and IgA than animalsadministered control lyophilized vegetative cells only.

FIG. 41 is a set of bar graphs showing anti-mLT IgA and IgGimmunoglobulin titer in murine serum samples and fecal samples 14 daysafter a third immunization or fourth immunization of animals. Theanimals were immunized sublingually with lyophilized B. subtilisvegetative cells and 5 μg of mLT adjuvant (LT 5+veg cells, SL; closedbars), or intranasally with lyophilized B. subtilis vegetative cells and10 μg of mLT adjuvant (LT 10+veg cells, IN; open bars).

FIG. 41 panel A shows that serum anti-mLT IgG was higher than anti-mLTIgA for both sublingual and intranasal immunization. The ratios ofsublingual IgG levels and intranasal IgG levels were comparable.

FIG. 41 panel B shows that fecal anti-mLT IgA amounts was higher thananti-mLT IgG for both sublingual and intranasal immunization.

FIG. 42 is a line graph showing log of serum titers for pigletsimmunized sublingually with lyophilized B. subtilis vegetative cellsBB3059 TetC (open squares); sublingually with lyophilized B. subtilisvegetative cells BB3059 TetC and mLT adjuvant (open triangles); orallywith lyophilized B. subtilis vegetative cells BB3059 expressing TetC(open circles); or sublingually with control lyophilized B. subtilisvegetative cells (closed diamonds). Animals immunized sublingually withlyophilized B. subtilis vegetative cells BB3059 expressing TetC with orwithout mLT adjuvant showed greater serum anti-TetC antibody titer thananimals immunized orally with either lyophilized B. subtilis vegetativecells BB3059 expressing TetC, or control lyophilized vegetative cells.Control lyophilized vegetative cells did induce antibody.

FIG. 43 is a line graph showing log of serum titer (ordinate) as afunction of time (abscissa) in mice immunized with dried lyophilizedvegetative cell vaccine expressing TetC according to immunizationschedules: biweekly (closed circles), monthly (open circles), orbimonthtly (closed triangle). The data show successful similar endpointsof high titer, with optimal antibody production at biweekly or monthlyschedules.

DETAILED DESCRIPTION

Infectious diseases remain a public health problem, in spite of theprogress in antibiotic and anti-viral chemotherapeutic agents. A classof viral diseases referred to as emerging diseases and exemplified bySARS and avian and/or swine influenza, have been causally associatedwith increased contact between wild animals that migrate, such as ducksand geese, with intensely farmed agricultural animals such as pigs, andfrom rapid global travel.

Spore-foaming microorganisms offer the possibilities of new classes ofvectors for administering one or more antigens of an infectious virus,in order to immunize human or animal subjects. Great variety in choiceof types of host cells enables the designer of the vaccine to use a cellgenotype that results in a single round of immunization, for example inhuman subjects, by using chromosomal markers that allow growth onlyunder highly restricted conditions, or by using a cell genotype chosento allow transmission from subject to subject, such as in a birdpopulation.

Most important, because bacterial and fungal spores remain viable undera very wide range of ambient environmental conditions, a spore-basedvaccine offers the possibility of storage at room temperatures ratherthan under refrigeration or freezing. See Acheson et al., U.S. Pat. No.5,800,821, issued Sep. 1, 1998, and incorporated herein by reference inits entirety.

Spores of bacterial genera within the group of streptomycetes aresufficiently heat resistant to survive extreme fluctuations of roomtemperature, for example substantial quantitative survival for at leasta few minutes at 50° C. Strains of the yeast Saccharomyces, a fungusthat produces ascospores, are resistant to several minutes of heat at60° C. See, Put et al., 1982, J. Appl. Bact. 52: 235-243. Similarly,spores of non-toxic strains of fungi, such as Penicillium strains thatare well known edible components of cheese (Roquefort, gorgonzola, etc.)and produce spores that may be used. Heat resistance for 10 minutes at50° C. was observed with spores from a variety of species of the fungusAspergillus (Pitt et al., 1970, Appl Microbiol. 20(5): 682-686).Genetics and recombinant techniques for many strains and species of bothstreptomycetes and fungi are well developed. See Hopwood et al.,Streptomyces, 1985, publ. John Innes Press; Kieser et al., PracticalStreptomyces Genetics, 2008, publ. John Innes Press.

However these spores are not so resistant to extreme conditions as arethe spores of Bacillus strains (which survive quantitatively even atsuch extreme conditions as boiling, and have been recovered as viablecolony forming units from insects preserved for millions of years inamber; Cano et al., 1995, Science 268:1060-1064). The methods herein aresuitable for use with spores of bacteria or fungi capable ofwithstanding ambient conditions of storage at room temperature.

The use of B. subtilis as a vehicle for vaccine antigen delivery is apromising new approach to mucosal immunization (Duc et al., 2003,Infect. Immun. 71: 2810-2818; Oggioni et al., 2003, Vaccine 21 Suppl. 2:S96-101). The primary model used to date has been the spore form of B.subtilis displaying tetanus toxin antigen on its surface. An advantageof using spores as vectors is that the spores are highly resistant toenvironmental stresses such as extremes of heat, pH, desiccation,freezing and thawing, and radiation (Nicholson et al., 2000, Microbiol.Mol. Biol. Rev. 64(3): 548-557). Heterologous antigens displayed on thespore surface as a fusion product with spore coat proteins have beenshown to elicit protective immune responses to tetanus toxin when sporesdisplaying tetanus toxin fragment C (TTFC) were given either orally orintranasally (Due et al., 2003, Infect. Immun. 71: 2810-2818). For oralimmunization, several rounds of high doses of spores (≧10¹⁰) werenecessary and the long-term immunogenic stability of these preparationshas not been rigorously tested. Moreover, the exposure of the antigen toproteases in the GI tract may reduce the availability of immunogenicprotein to the GI immune system (Duc et al. 2003).

Orally administered spores of B. subtilis survive passage through thegastrointestinal tract of mice and may germinate in the intestines toyield replicative vegetative cells; the intestinal tract becomes brieflycolonized (Spinosa et al., 2000, Res. Microbiol. 151: 361-368; Casula etal., 2002, Appl. Environ. Microbiol. 68: 2344-2352). If spores weredesigned to generate antigen only after germination in the intestinaltract, such a spore-based vaccine would address the issues of antigendegradation during storage and during passage through the GI tract andwould potentially be stable indefinitely. The B. subtilis spore-basedvaccine induces a serum antibody response to Yersinia pseudotuberculosisinvasin by spores engineered to display invasin on the vegetative cellsurface after germination and outgrowth (Acheson et al., U.S. Pat. No.5,800,821, issued Sep. 1, 1998). Oral inoculation with B. subtilisspores engineered to express TTFC after germination in the vegetativecell cytoplasm was shown to induce protective antibodies (Uyen et al.,2007, Vaccine 25(2): 356-365). It is not known how well engineeredstrains of B. subtilis colonize the intestine of humans or if therewould be interference of colonization from other intestinal micoflora.

Because of the lack of immune responses found by some to live bacterialvectors given orally, another approach to mucosal immunization is theintranasal route. Attenuated Salmonella typhi expressing TTFC elicitedprotective immunity to tetanus toxin after the vaccine was administeredintranasally, but not orally (Galen et al., 1997, Vaccine 15(6-7):700-708). Use of attenuated pathogenic bacteria as vectors has thegeneral disadvantage that sufficient attenuation of virulence isrequired to assure safety. For this reason, bacteria that are generallyregarded as safe are preferable. For instance, Lactobacillus plantarumexpressing TTFC was shown to protect against tetanus toxin challengeafter intranasal administration (Grangette et al., 2001, Infect. Immun.69(3):1547-1553). Like lactobacilli, B. subtilis is also generallyregarded as safe, and is neither pathogenic nor toxigenic to humans,animals, or plants (Sonenshein et al 1993). B. subtilis has beenextensively studied as a model gram-positive bacterium, and isadvantageous for genetic manipulation. Stable genetically engineeredconstructs can be integrated into the bacterial chromosome, making it agood candidate for vaccine preparation.

Bacterial genera such as Bacillus and others that produce spores, andfungal species are within the scope of embodiments of the methods andcompositions herein, if they satisfy criteria of suitability forengineering vaccines, viz., production of stable spores, andnon-toxicity to animals of spores and vegetative cells. For example,cells of non-toxic streptomycete strains such as Streptomyces lividans,S. coelicolor, and S. reticuli may be engineered by conventional genetictechniques to express cytoplasmically an antigen encoded by the genomeof an infectious agent, such that the antigen is synthesized in solubleform, during vegetative growth of the cells. The antigen while made as asoluble material becomes associated with spores during the sporulationprocess. The spores are prepared by conventional techniques into avaccine composition, and when administered to a subject results in animmune response capable of protecting the subject from infection by theinfectious agent that is the source of the antigen.

Compositions including Bacillus spores and vegetative cells expressingantigen are administered to the subject, for example by contacting themucosal tissues. Mucosal tissues or mucosae or mucosal cells areepithelial linings of tissue that function to modulate absorption intoand secretion from of nutrients and signaling macromolecules to and fromunderlying organs and tissues. Mucosal tissues are found for examplelining nose, throat, mouth, eye, eyelid, esophagus, female genitalia,male genitalia, anus, urinary tract, and digestive tract.

The term, “antigen” as used herein and in the claims refers to a proteinor a portion of a protein, isolated from nature or synthesized, orexpressed in and purified from a recombinant cell, or a peptide, or aderivatized version thereof containing one or a few additional aminoacids, including sequences of amino acids that are of biological origin,or are not found in nature. The antigen is a peptide of sufficientlength to provoke an immune response in an animal having an immunesystem, generally at least about 4 to 7 amino acids in length.

The term “derivative” as used herein and in the claims may be a protein,peptide, or chemically related foam of that protein having an additionalsubstituent on an amino acid, for example, N-carboxyanhydride group, aγ-benzyl group, an ε,N-trifluoroacetyl group, or a halide group attachedto an atom of the amino acid of a protein.

The term, “immune response” means any natural function of an immunesystem, and includes without limitation, any cellular function or organfunction characteristic of ability to distinguish a component of selfand non-self by an organism, for example, ability to bind to an MHCclass I or class II protein, or ability to bind to a cell having suchproteins, ability to activate killer T cells, or to elicit production ofantibodies of any type are all within the definition.

Immunizations have been well-known in the medical arts since discoveryof cowpox vaccination over two hundred years ago, and include injectionby a variety of routes including subcutaneous, and oral delivery, e.g.,of Sabin polio vaccine. Discovery herein of efficiency of a spore-basedvaccine delivered by an intranasal route is an important and surprisingresult.

The term, “associated” as used herein and in the claims refers to thephysical relationship of an antigen to a spore, or a vegetative cell orportion thereof. During sporulation, the antigen is produced duringvegetative phase of cell growth and is physically associated with thespore. The antigen may be displayed on the spore's surface, or may beentrained within the spore coat, and the ability of the spores orvegetative cells or portions thereof to elicit an immune response doesnot depend on any particular physical location of the antigen within thespore or cell, nor any mechanism of packaging. In contrast toembodiments of the methods and compositions herein, in which the antigenis associated with spores and is not necessarily covalently bound to anyparticular spore component, Acheson et al., issued Sep. 1, 1998, U.S.Pat. No. 5,800,821 shows a vaccine using spores engineered bygenetically manipulating spore-forming bacterial cells to contain acertain DNA sequence encoding an antigen, producing spores produced as agenetic fusion to spore surface proteins during the sporulation phase inthe cells.

The term, “adjuvant” as used herein and in the claims refers to anycompound which when administered together with an antigennonspecifically enhances the immune response to that antigen. Adjuvantsmay be insoluble and undegradable substances (e.g., inorganic gels suchas aluminum hydroxide), or water-in-oil emulsions such as incompleteFreund's adjuvant. Generally, adjuvants retard the destruction ofantigen and allow the persistence of low but effective levels of antigenin the tissues and also nonspecifically activate the lymphoid system byprovoking an inflammatory response. One of the most effective adjuvantsis Freund's complete adjuvant having mycobacteria suspended in awater-in-oil emulsion, however the intense inflammatory response itprovokes precludes its clinical use. Examples herein show enhancedimmune responses with several adjuvants including cholera toxin and anon-toxic Escherichia coli heat-labile enterotoxin variant, which areadministered with spores.

Both cholera toxin produced by Vibrio cholerae and Escherichia coli heatlabile enterotoxin have been widely used as adjuvants in experimentalsystems, and each induces significant antibody responses to adjuvantsand are also potent mucosal adjuvants for co-administrated, unrelatedantigens, especially when give by the oral route. Cholera toxinfunctions as an adjuvant by inducing antigen specific CD4⁺ T cells tosecrete interleukin 4 (IL-4), IL-5, IL-6, and IL-10. Heat labileenterotoxin produced by some enterotoxigenic strains of Escherichia colifunctions as an adjuvant by inducing Th1 and Th2 cytokine responses.Both cholera toxin produced by Vibrio cholerae and heat labileenterotoxin produced by Escherichia coli are multi-subunitmacromolecules composed of two structurally, functionally, andimmunologically separate A and B subunits (Yamamoto et al., 1997, Proc.Natl. Acad. Sci. USA 94(10): 5267-5272).

The term “thermally-stable” as used herein and in the claims relates toan enhanced persistence of an active substance or pharmaceutical productas a function of time under the influence of a variety of environmentalfactors, primarily temperature, and is also affected by other conditionssuch as humidity and light in comparison with a control preparation thatis not thermally stable. In making this determination, product-relatedfactors also influence the stability, e.g., the chemical and physicalproperties of the active substance and the pharmaceutical excipients,the dosage form and its composition, the manufacturing process, thenature of the container-closure system, and the properties of thepackaging materials. Also, the stability of excipients that may containor form reactive degradation products are considered.

Stress testing of the active substance is used to identify the potentialdegradation products, and to establish degradation pathways andstability of the molecule. The nature of the stress testing depends onthe individual active substance and the type of pharmaceutical productinvolved.

In general, an active substance is evaluated under storage conditions(with appropriate tolerances) that test thermal stability and, ifapplicable, sensitivity to moisture. The storage conditions and thelengths of time period for study are appropriate to consider storage,shipment, and subsequent use appropriate to the climatic zone or zonesin which the active substance is likely intended to be stored. Long-termtesting extends for a period about a month, or, about three months,about six months, or about twelve months. Testing includes a number ofproduct batches in conditions of packaging and temperatures that arerepresentative of the product's intended use.

Data from an “accelerated” storage condition, if appropriate, areobtained to test the product at conditions beyond those intended, i.e.,excessively high or low temperatures compared to potential actualambient conditions. An accelerated storage condition includes tests ofconditions that mimic handling issues such as prolonged exposure toexcess moisture and variable volume delivery of a composition includingthe active substance. Calculations of the data obtained underaccelerated conditions are then used to extrapolate the presumedstability of the product in normal environments and conditions, althoughthese calculations are an estimate of stability of the product undernormal conditions. Data obtained from accelerated storage conditions arecombined with other data including long-term testing described above todetermine the stability of the product.

U.S. Pat. No. 6,187,319 (Herrmann et al., issued Feb. 13, 2001)describes a method of producing an immune response in an animal to arotavirus antigen, by administering an isolated rotavirus VP6polypeptide of a different strain of rotavirus to produce an effectiveimmune response. The VP6 polypeptide was delivered directly, or by usinga DNA plasmid or a virus to express the polypeptide in the recipient,with transcriptional and translational regulatory sequences encoded bythe plasmid or virus. U.S. Pat. No. 6,165,993 (Herrmann et al., issuedDec. 26, 2000) describes a method of eliciting an immune response orprotective immunity with a vaccine having DNA encoding an antigen (e.g.,capsid proteins or polypeptides of a rotavirus such as VP4, VP6 andVP7), the antigen encoded by a nucleotide sequence in a plasmid vector.

A feature provided by the present invention herein is a method ofimmunizing a subject to an infectious agent, the method including stepsof: sporulating a vegetative host bacterial cell which contains anisolated nucleotide sequence encoding an antigen of the infectiousagent, such that the nucleotide sequence is operably linked to apromoter for cytoplasmic vegetative expression of the antigen, such thatthe spores are associated with the antigen and, contacting the subjectwith a composition including the spores, such that the antigen immunizesthe subject to the infectious agent. In general, the infectious agent isviral or bacterial. For example, the infectious agent is at least onebacterium selected from the group of consisting of Bacillus anthracis,Clostridium tetani, Corynebacterium diphtheriae, Bordetella pertussis,Mycobacterium tuberculosis, Salmonella typhimurium, Staphylococcusaureus, Streptococcus pneumoniae, Treponema pallidum, Neisseriagonorrhoeae, and the like. For example, the infectious agent is at leastone virus selected from the group consisting of human immunodeficiencyvirus (HIV), influenza, polio, herpes, smallpox, measles, mumps,rubella, rotavirus, chicken pox, rabies, West Nile virus, eastern equineencephalitis, norovirus, and the like. An exemplary antigen is arotavirus antigen.

The method in related embodiments further includes prior to sporulating,obtaining the isolated nucleotide sequence encoding the rotavirusantigen from a rotavirus strain that is bovine or murine. For example,the rotavirus antigen is a viral virion protein, for example, the viralvirion protein is selected from at least one of the group consisting ofVP2, VP4, VP6, VP7, NSP4, and a portion or a derivative thereof.

In general, the subject is a vertebrate animal. For example, thevertebrate animal is from at least one of the group of an agriculturalanimal, a high value zoo animal, a research animal, a human, and a wildanimal found in a densely populated human environment such as a wildbird.

The method in related embodiments further includes contacting thesubject by administering the composition by a route selected from atleast one of intravenous, intramuscular, intraperitoneal, intradermal,mucosal, and subcutaneous routes. For example, contacting the subject isby intranasal administration. For example, the intranasal administrationincludes inhalation or nose drops. Inhalation methods include use of anebulizer or an atomizer, and include a measured dose.

In general, the vegetative host bacterial cell is a Bacillus cell. Forexample, the Bacillus is Bacillus subtilis, although other species ofbacilli and other spore-forming organisms are also within the scope ofthe methods herein.

The composition used in the method herein includes in relatedembodiments an adjuvant, for example, the adjuvant is selected from atleast one of the group consisting of cholera toxin, a non-toxic variantof Escherichia coli labile toxin, and a portion or a derivative thereof.

The method according to related embodiments further involves observingresistance of the composition to at least one condition selected fromthe group of heat, drying, freezing, deleterious chemicals andradiation.

An embodiment of the method further involves measuring an antibody titerin serum of an infected subject, such that increase in antibody for theantigen in comparison to a control serum is an indication of efficacy ofthe immunogenicity of the composition. Suitable control sera includepre-immune serum from the subject, or serum from a different subjectreceiving a different antigen. Still another feature of the inventionprovided herein is measuring an amount of viral shedding in the subjectafflicted by the infectious agent, such that a decrease in fecal viruscompared to that in a control subject also afflicted by the infectiousagent and not contacted with the composition, is a measure of efficacyof the immunogenicity of the composition.

A featured embodiment of the invention provided herein is athermally-stable vaccine composition for immunizing a subject with anantigen from an infectious agent, the composition including spores froma Bacillus cell that contains an isolated nucleotide sequence encodingthe antigen, the nucleotide sequence being genetically engineered andhaving been integrated into the host bacterial chromosome or carried ona plasmid and provided with appropriate transcriptional andtranslational regulatory sequences, such that the cell expresses theantigen cytoplasmically as a soluble component during vegetative growth,and upon sporulation by the cell, the antigen is associated with thespores, and the composition comprising the spores is effective toimmunize the subject. For example, the antigen is a viral protein or aportion or a derivative thereof. For example, the viral protein is aviral virion protein. For example, the viral virion protein is selectedfrom at least one of the group consisting of VP2, VP4, VP6, VP7, NSP4,and a portion or a derivative thereof. An exemplary, Bacillus isBacillus subtilis.

The composition in related embodiments includes an adjuvant. Forexample, the adjuvant is selected from at least one of the groupconsisting of cholera toxin, a non-toxic variant of Escherichia colilabile toxin, and a portion or a derivative thereof.

In related embodiment of the composition, the isolated nucleotidesequence encoding the antigen is from a strain that is bovine or murine.

The invention herein also features a vaccination kit that includes aunit dose of the composition according to any of the above embodiments,a container, and instructions for use. In related embodiments, theinstructions include storage at a room temperature of from about 4° C.to about 45° C. and the like (calculation of 45° C. is that thistemperature is the same as 113° F.).

Pharmaceutical Compositions

An aspect of the present invention provides pharmaceutical compositions,wherein these compositions comprise spores associated with an antigenfrom an infectious agent, and optionally further include an adjuvant,and optionally further include a pharmaceutically acceptable carrier.

In certain embodiments, these compositions optionally further compriseone or more additional therapeutic agents. In certain embodiments, theadditional therapeutic agent or agents are selected from the groupconsisting of growth factors, anti-inflammatory agents, vasopressoragents, collagenase inhibitors, topical steroids, matrixmetalloproteinase inhibitors, ascorbates, angiotensin II, angiotensinIII, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin,transforming growth factors (TGF), keratinocyte growth factor (KGF),fibroblast growth factor (FGF), insulin-like growth factors (IGF),epidermal growth factor (EGF), platelet derived growth factor (PDGF),neu differentiation factor (NDF), hepatocyte growth factor (HGF), andhyaluronic acid.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa.,1995 discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Carriersare selected to prolong dwell time for example following inhalation orother form of intranasal administration, or other route ofadministration.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, sugars such asglucose, and sucrose; starches such as corn starch and potato starch;cellulose and its derivatives such as sodium carboxymethyl cellulose,ethyl cellulose, and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients such as cocoa butter and suppository waxes;oils such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil, and soybean oil; glycols such as propylene glycol;esters such as ethyl oleate and ethyl laurate; agar; buffering agentssuch as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

In yet another aspect, according to the methods of treatment of thepresent invention, the immunization is promoted by contacting the animalwith a pharmaceutical composition, as described herein. Thus, theinvention provides methods for immunization comprising administering atherapeutically effective amount of a pharmaceutical compositioncomprising active agents that include a spore preparation having anassociated antigen from an infectious agent, to a subject in needthereof, in such amounts and for such time as is necessary to achievethe desired result. It will be appreciated that this encompassesadministering an inventive vaccine as described herein, as a preventiveor therapeutic measure to promote immunity to the infectious agent, tominimize complications associated with the slow development of immunity(especially in compromised patients such as those who are nutritionallychallenged, or at risk patients such as the elderly or infants).

In certain embodiments of the present invention a “therapeuticallyeffective amount” of the pharmaceutical composition is that amounteffective for promoting appearance of antibodies in serum specific forthe chosen antigen, or disappearance of disease symptoms, such as amountof virus in feces or in bodily fluids or in other secreted products. Thecompositions, according to the method of the present invention, may beadministered using any amount and any route of administration effectivefor generating an antibody response. Thus, the expression “amounteffective for promoting immunity”, as used herein, refers to asufficient amount of composition to result in antibody production orremediation of a disease symptom.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active agent(s) or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state, e.g., exposure to infectious agent in thepast or potential future exposure, or exposure to a seasonal allergen;age, weight and gender of the patient; diet, time and frequency ofadministration; drug combinations; reaction sensitivities; andtolerance/response to therapy. Long acting pharmaceutical compositionsmight be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularcomposition.

The active agents of the invention are preferably formulated in dosageunit form for ease of administration and uniformity of dosage. Theexpression “dosage unit form” as used herein refers to a physicallydiscrete unit of active agent appropriate for one dose to beadministered to the patient to be treated. It will be understood,however, that the total daily usage of the compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. For any active agent, the therapeuticallyeffective dose can be estimated initially either in cell culture assaysor in animal models, usually mice, rabbits, dogs, or pigs. The animalmodel is also used to achieve a desirable concentration range and routeof administration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of active agentwhich ameliorates at least one symptom or condition. Therapeuticefficacy and toxicity of active agents can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose is therapeutically effective in 50% of thepopulation) and LD50 (the dose is lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD50/ED50. Pharmaceutical compositionswhich exhibit large therapeutic indices are preferred. The data obtainedfrom cell culture assays and animal studies is used in formulating arange of dosage for human use.

The therapeutic dose shown in examples herein is at least about 10⁸,about 3×10⁸, about 10⁹, or at least about 3×10⁹ spores/dose/animal. Asbacterial spores are readily produced and are inexpensively engineeredand designed and stored, greater doses for large animals areeconomically feasible. For an animal several orders of magnitude largerthan experimental animals used in examples herein, the dose is easilyadjusted, for example, to about 3×10¹⁰, about 3×10¹¹, to 3×10¹² or about3×10¹³, for animals such as humans and small agricultural animals.However doses of about 3×10¹⁴, 3×10¹⁵, or even about 3×10¹⁶, or about3×10¹⁷, for example for a high value zoo animal or agricultural animalsuch as an elephant, are within the scope of the invention. Forpreventive immunizations, or periodic treatment, or treatment of a smallwild animal, smaller doses such as less than about 3×10⁹, or less thanabout 3×10⁸, or even less than about 3×10⁷ per dose, are within thescope of the invention.

Administration of Pharmaceutical Compositions

After formulation with an appropriate pharmaceutically acceptablecarrier in a desired dosage, the pharmaceutical compositions of thisinvention can be administered to humans and other mammals topically (asby powders, ointments, or drops), orally, rectally, mucosally,sublingually, parenterally, intracisternally, intravaginally,intraperitoneally, bucally, ocularly, or nasally, depending on theseverity and nature of the infectious agent being treated.

In various embodiments of the invention herein, oral and intranasalinoculation using spores and vegetative cells of B. subtilis engineeredto express TTFC are compared. It was observed that high titers ofantibodies, sufficient for protection against a lethal dose of tetanustoxin, were produced in mice after intranasal administration ofvegetative cells expressing cytoplasmic TTFC or spores displaying TTFCas a fusion protein to a spore coat protein. These vaccines proved tohave a long shelf life at elevated temperatures when stored in the drystate.

While intranasal administration was demonstrated to be surprisinglyeffective in examples herein, the antigen associated with spores and/orvegetative cells, preferably lyophilized, following vegetativecytoplasmic expression of the antigen prior to sporulation leads to avaccine that is administered in any of a variety of routes. For example,it is envisioned that for agricultural animals, such as immunizingchicken or ducks for viral influenza, oral or intranasal administrationwould be highly suitable.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active agent(s), theliquid dosage forms may contain inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The activeagent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. For example, ocular or cutaneous infections may be treatedwith aqueous drops, a mist, an emulsion, or a cream. Administration maybe therapeutic or it may be prophylactic. Prophylactic formulations maybe present or applied to the site of entry of potential diseaseorganisms, such as, in the case of topical infectious organisms such asherpes virus, to wounds, such as contact lenses, contact lens cleaningand rinsing solutions, containers for contact lens storage or transport,devices for contact lens handling, eye drops, surgical irrigationsolutions, ear drops, eye patches, and cosmetics for the eye area,including creams, lotions, mascara, eyeliner, and eyeshadow. Theinvention includes products which contain the compositions having thelyophilized vegetative cells or spores (e.g., gauze bandages or strips),and methods of making or using such devices or products. These devicesmay be coated with, impregnated with, bonded to or otherwise treatedwith a vaccine composition. For sites of disease entry that areprimarily spread by droplet infection, such as rhinovirus and influenza,intranasal administration is suitable.

The ointments, pastes, creams, and gels may contain, in addition to anactive agent of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the agents of thisinvention, excipients such as talc, silicic acid, aluminum hydroxide,calcium silicates, polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of the active ingredients to the body. Such dosage forms can bemade by suspending spores in the matrix applied to the patches, ordispensing the compound in the proper medium. Absorption enhancers canalso be used to increase the flux of the antigenic peptide released fromspores into the compound, for passage across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized prior to addition of spores,for example, by filtration through a bacterial-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium prior to use. In order to prolong theeffect of an active agent, it is often desirable to slow the absorptionof the agent from subcutaneous or intramuscular injection. Delayedabsorption of a parenterally administered active agent may beaccomplished by dissolving or suspending the agent in an oil vehicle.Injectable depot forms are made by forming microencapsule matrices ofthe agent in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of active agent to polymer and the nature ofthe particular polymer employed, the rate of active agent release can becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsare also prepared by entrapping the agent in liposomes or microemulsionswhich are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the active agent(s) ofthis invention with suitable non-irritating excipients or carriers suchas cocoa butter, polyethylene glycol or a suppository wax which aresolid at ambient temperature but liquid at body temperature andtherefore melt in the rectum or vaginal cavity and release the activeagent(s).

Solid dosage forms for oral, mucosal or sublingual administrationinclude capsules, tablets, pills, powders, and granules. In such soliddosage forms, the active agent is mixed with at least one inert,pharmaceutically acceptable excipient or carrier such as sodium citrateor dicalcium phosphate and/or a) fillers or extenders such as starches,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as milksugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings, release controlling coatings and other coatings well known inthe pharmaceutical formulating art. In such solid dosage forms theactive agent(s) may be admixed with at least one inert diluent such assucrose or starch. Such dosage forms may also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,tableting lubricants and other tableting aids such a magnesium stearateand microcrystalline cellulose. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. They mayoptionally contain opacifying agents and can also be of a compositionthat they release the active agent(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes.

Uses of Pharmaceutical Compositions

As discussed above and described in greater detail in the Examples,spores, spore preparations, vegetative cells, and vegetative cellpreparations, e.g., by lyophilization, particularly bacterial spores,e.g., spores of a Bacillus, are used to prepare a heat resistant stableactive vaccine by associating with an antigen from an infectious agentduring sporulation. In general, it is believed that these vaccines willbe clinically useful in immunizing subjects for resistance to infectiousdiseases. The present invention encompasses the treatment of a varietyof infectious diseases arising from infection with bacteria, viruses,fungi, and parasites. The vaccines herein are particularly useful totreat compromised patients, particularly those anticipating therapyinvolving, for example, immunosuppression and complications associatedwith systemic treatment with steroids, radiation therapy, non-steroidalanti-inflammatory drugs (NSAID), anti-neoplastic drugs andanti-metabolites.

In addition, vegetative cells and spores associated with antigens fromtumors, tumor cells/cell lines, and allergy producing proteins arecontemplated. Allergy producing proteins and macromolecules include forexample dust mite proteases, cat and dog salivary proteases, andproteins found in pollens of allergens such as pollen of grass and treessuch as, ragweed, timothy and maple trees. The unprecedented stabilityof the vaccine compositions, and the rapid response as shown byappearance of serum antibodies following intranasal administration,indicates that the vaccines can be used by patients in a home setting,and can be supplied in suitable single dose or measured dose formats, tobe used as needed, for example, seasonally.

A skilled person will recognize that many suitable variations of themethods may be substituted for or used in addition to those describedabove and in the claims. It should be understood that the implementationof other variations and modifications of the embodiments of theinvention and its various aspects will be apparent to one skilled in theart, and that the invention is not limited by the specific embodimentsdescribed herein and in the claims. The present application mentionsvarious patents, scientific articles, and other publications, each ofwhich is hereby incorporated herein in its entirety by reference.

The invention having how been fully described, it is exemplified by thefollowing examples and claims which are for illustrative purpose onlyand are not meant to be further limiting.

EXAMPLES Example 1 Genetic Engineering of Rotavirus VP6 and ChromosomalInsertion

VP6 is the inner capsid protein of rotavirus and has a molecular weightof approximately 45 kd, and is a known immunogen for use in vaccines totreat rotaviral infection. Fusions of the bovine and murine VP6 codingregions to a version of the Pspac promoter (Yansura et al., 1984, Proc.Natl. Acad. Sci. USA 81:439-443) were integrated at the sacA locus ofthe B. subtilis chromosome. The Pspac promoter was altered byintroducing two site-directed mutations, C-12T and A+1G. These mutationsincreased the strength of the promoter. The VP6 coding regions wererecovered from pCR.2.1-VP6 bovine (obtained from Dr. L. J. Saif, OhioState University, Wooster, Ohio) and pBluescript-VP6 murine (GenBankaccession no. U36474; obtained from Dr. H. B. Greenberg, StanfordUniversity, Palo Alto, Calif.) by PCR and were cloned initially invector pBB1378. The latter plasmid carries the B. subtilis veg promoterand a kanamycin resistance gene surrounded by the 5′ and 3′ ends of thesacA locus. An appropriately placed ribosome binding site wasincorporated into VP6 constructs during the PCR step. The relevantfragments containing the ribosome binding site and the VP6 codingsequences were excised from the resulting plasmids by treatment withPacI and SacI and were cloned in similarly digested pBB1375. The latterplasmid carries the mutant Pspac promoter and a kanamycin resistancegene surrounded by the 5′ and 3′ ends of the sacA locus. When introducedinto competent cells of B. subtilis strain BB2534 [ΔthyA ΔthyB trpC2ΔsacA::(thyA⁺ cat)], the resulting transformants, BB2543 for bovine VP6and BB2547 for murine VP6, arose by double crossover recombination atthe sacA locus. B. subtilis strains BB2666 (containing bovine VP6) andBB2667 (containing murine VP6) are Thy⁺ versions of the latter strains,respectively. The control strain, BB2643, had the same geneticorganization as BB2666 and BB2667, except that the VP6 coding sequencewas absent.

Example 2 Growth of Bacteria and Preparation of Vegetative Cells andSpores

Bacillus subtilis type 168 strain was used to construct the recombinantstrains expressing TTFC. E. coli strain JM107 was used for cloningexperiments. Bacterial strains were routinely grown in Luria broth (LB)and plates containing solid LB medium were prepared with neomycin (5μg/ml) for B. subtilis, or kanamycin (25 μg/ml) or ampicillin (100μg/ml) for E. coli. For some experiments, B. subtilis cells were grownin a defined medium (TSS) supplemented with glucose (0.5-1%), ammoniumchloride (0.2%) and sodium glutamate (0.2%).

B. subtilis strains grown overnight on L agar plates were used toinoculate 4-L cultures in DS medium (Fouet et al., 1990; J. Bacteriol.172: 835-844). After incubation with shaking (200 rpm) at 37° C. for 48hrs, the mixture of spores and non-sporulating bacteria was harvested bycentrifugation, washed with sterile deionized water, treated with eggwhite lysozyme (1 mg/ml) to kill non-sporulating cells, washed fiveadditional times with sterile deionized water and stored at 4° C. insterile water. Spores were titered by direct counting using aPetroff-Hauser chamber and by comparing colony-forming ability beforeand after heating a sample to 80° C. for 10 min.

The following bacterial strains were used in the examples: control B.subtilis spores (not carrying genes encoding antigens for vaccines), andB. subtilis capable of displaying either bovine or murine-derived VP6(e.g., bovine (Bo)VP6 or murine (Mu) VP6 as described herein).

Example 3 Administration and Response Testing

BALB/c female mice of about 4-6 weeks were immunized intranasally withspores according to a specific schedule (e.g., dose administered at 0,14, and 28 days) as shown in the figures. Each animal was administered3×10⁹ spores per dose.

Adjuvant was used with an antigen in examples herein to co-immunizeanimals to enhance the immune response. Cholera toxin (CT) produced byvarious strains of Vibrio cholerae promotes Th2 cytokine responses, andimproves efficacy of the immune response involving one or more of IgG1,IgE, and mucosal IgA antibodies. Escherichia coli LT (R192G) is amutated variant of a heat-labile enterotoxin produced by enterotoxigenicstrains of E. coli. The LT (R192G) variant is non-toxic, and induces Th1and Th2 cytokine responses and improves efficacy of the immune responseinvolving one or more of IgG1, IgG2a, IgG2b, and mucosal IgA antibodies.Adjuvant volumes of 20 μl were used for immunization; CT wasadministered at 10 μg/dose, and E. coli LT (R192G) was administered at 5μg/dose or 10 μg/dose.

Methods to determine effectiveness of immunization of mice administeredwith VP6 spore preparations (bovine or murine-derived VP6 spores or anegative control) included: mice were sampled for titer of serumanti-VP6 antibody, which was measured using ELISA. Mice were challengedorally with the agent that causes epizootic diarrhea of infant mice(EDIM), rotavirus, and were monitored for the course of rotavirusinfection by measuring appearance of virus VP6 antigen in feces.

Example 4 Immunization with Bovine or Murine-Derived VP6 SporePreparations in Presence of CT Caused Increased Serum Anti-VP6 AntibodyTiter

Animals were immunized with VP6 spores (with or without CT) and weretested for appearance of anti-VP6 antibody in serum. The mice wereimmunized intranasally on days 0, 14, and 28 and were tested on days 14,28 and 42.

Animals immunized with spore preparations associated with bovine- ormurine VP6 (FIG. 1, triangles or circles) showed significant productionin serum of anti-VP6 as measured by titer response at day 28 and day 42compared to animals administered control VP6 spores. Murine-derived VP6spore preparations showed a slightly greater ability to elicit anantibody titer response than bovine-derived VP6 at day 42, and bovinederived VP6 spores resulted in a slightly greater response at day 28.See FIG. 1. Animals immunized with spore preparations of BB2643, a B.subtilis strain that does not contain rotavirus-derived sequences (withor without CT; squares) produced almost no serum anti-VP6 ELISA titerresponse at any of the tested dates.

Immunizing animals with bovine- or murine-associated VP6 sporepreparations with CT adjuvant increased the serum anti-VP6 titerresponse compared to immunizing with bovine or murine-derived VP6 sporepreparations absent adjuvant, at each of day 28 and day 42. (FIG. 1,open) The relative increase for serum anti-VP6 titer response forbovine-associated VP6 spore preparations with CT compared to controlspore preparations absent adjuvant was similar at day 28 and day 42.

Intranasal immunizations with bovine- or murine-associated VP6 sporepreparations caused increased serum antibody titer compared to controlspore preparations not displaying VP6. Also, use of an adjuvant, i.e.,administering bovine- or murine-associated VP6 spore preparations withCT, increased the serum antibody titer even further (open circles, opentriangles).

Example 5 Intranasal Immunization with Bovine- or Murine-Associated VP6Spores with CT Reduced Amount of Rotavirus in Feces in EDIM RotavirusInfection Model

Animals immunized with VP6 spore preparations were challenged orallywith rotavirus, using the EDIM animal model of rotavirus infection, andmice were tested for infection by measuring viral antigen in feces byELISA each day for seven days.

Immunizing animals with VP6-associated spore preparations with orwithout adjuvant CT resulted in almost complete suppression of thedisease. Animals administered control spores showed massive viralproduction from days 2 to 6 (FIG. 2). Bovine- or murine-associated VP6spore preparations with CT adjuvant resulted in substantially reducedviral presence in comparison to administration of control sporepreparations (with or without CT) with the infection appearing only atday 2, or at reduced levels from days 2 to 6. Animals administeredmurine-associated VP6 spore preparations and CT adjuvant showedsubstantially no virus in feces on day 3 (less than 0.2 OD). Incontrast, animals administered control spore preparations continued toproduce massive amounts of virus through day 6.

These data show that intranasal immunizations with VP6-associated sporepreparations with CT caused reduced rotavirus infection after EDIMrotavirus challenge. This result is surprising because the VP6 antigenassociated with the spores was expressed during vegetative growth of thecells, and as a cytoplasmically soluble product, rather than as a fusionto a sporulation protein during the sporulation phase.

Animals immunized with bovine- or murine-associated VP6 spores showedeffective immunization: data show increased serum anti-VP6 antibodytiter as measured by ELISA and reduced fecal rotavirus antigen inrotavirus challenged mice (FIG. 1 and FIG. 2, respectively). The use ofadjuvant CT with bovine- or murine-associated VP6 spore preparationsfurther improved immune response in mice, as shown by the increasedserum anti-VP6 ELISA titer (100-fold increase) and reduced rotaviruspresence (3-fold to 10-fold) compared to control administered sporepreparations in absence of adjuvant.

To test the effect of adjuvant alone, groups of animals wereadministered CT with control spore preparations, and the data show noappearance of serum anti-VP6 antibody, or reduction of rotaviruspresence in EDIM animals (FIGS. 1 and 2).

Example 6 Intranasal Immunizations with Bovine- or Murine-Associated VP6Spores with LT (R192G) Show Increased Serum Anti-VP6 Titer Response

Animals were administered VP6-associated spore preparations withadjuvant LT (R192G), and were tested for serum anti-VP6 antibody titer.The animals were administered spore preparations intranasally on days 0,14, and 28 and serum was obtained on days 14, 28 and 42.

It was observed that animals immunized with bovine-associated VP6 sporeswith 5 μg/dose or 10 μg/dose LT (R192G) produced a substantial titer ofantibody (see FIG. 3). Bovine-associated VP6 spore preparations withadjuvant LT (R192G) resulted in greater serum antibody titers torotavirus VP6, than murine-associated VP6 spores absent adjuvant (FIGS.1 and 3). Both amounts of LT (R192G) of 5 μg/dose or of 10 μg/dose withthe bovine- or murine-associated VP6 spores was effective in increasingserum antibody response to rotavirus VP6.

Control spore preparations without antigen with 10 μg/dose LT (R192G)produced no serum anti-VP6 ELISA titer response at all time periods,similar to the results observed with animals immunized with controlspore preparations with 10 μg/dose CT in FIG. 1, as expected. These datashows that adjuvant enhances immune responses to spore preparationscontaining rotavirus VP6.

Example 7 Intranasal Immunizations with Bovine- or Murine-Associated VP6Spore Preparations with LT (R192G) Show Reduced Rotavirus Infection inan EDIM Disease Model

Animals were immunized intranasally with VP6-associated sporepreparations, challenged orally with EDIM rotavirus, and were tested forviral production in feces, by ELISA for presence of the VP6 antigen, foreach of seven days.

Animals immunized with murine-associated VP6 spore preparations with 10μg/dose LT (R192G), bovine-associated VP6 with μg/dose LT (R192G), orbovine-associated VP6 with 5 μg/dose LT (R192G) showed substantiallyreduced disease symptoms (FIG. 4, open symbols). The animalsadministered bovine- or murine-associated VP6 spores preparations showedalmost no viral antigen in feces nor did animals administered bovine VP6spore-associated preparations with adjuvant LT (R192G) at 5 μg/dose or10 μg/dose (FIG. 4).

These data show that immunizing intranasally with bovine or murinederived VP6 spores preparations with LT (R192G) substantially reducedrotavirus in feces.

Animals administered bovine- or murine-associated VP6 sporespreparations with LT (192G) were effectively immunized, as demonstratedby the increased serum anti-VP6 ELISA titers and the reduced fecalrotavirus content in EDIM rotavirus challenged animals.

B. subtilis spore preparations associated with a viral antigen wereeffective vaccines, generating protective immunity against rotaviruschallenge in the EDIM animal disease model. Animals immunized withbovine- or murine-associated VP6 spores had significantly increasedserum anti-VP6 response titers as compared to control spores, and anadjuvant such as CT or LT (R192G) further increased the serum antibodyresponse to rotavirus VP6.

Example 8 Recombinant Strains Expressing TTFC

To construct a recombinant strain that expresses TTFC during vegetativegrowth stage, vector pBB1375 for expression of cloned DNA under thecontrol of a highly active version of the semi-synthetic spac promoterwas constructed by site-directed mutagenesis. Plasmid pBB1375 wasderived from pSac-Kan (Middleton et al., 2004, Plasmid 51:238-245) bydeleting the BseRII fragment (resulting in pBB1364) and then introducingPspac between the BglII and XbaI sites. The version of the spac promoterin pBB1427 has two single-nucleotide mutations (SEQ ID No: 1) inconformance with the consensus sequences for promoters recognized by thesigma-A form of B. subtilis RNA polymerase (FIG. 5). The ribosomebinding site (RBS) and ATG initiation codon of the B. subtilis gsiB genewere inserted between the spac promoter and tetC. The tetC sequence frompositions 2855 to 4237 of the tetanus toxin gene of Clostridium tetani(GenBank no. X04436) were amplified and fused to the ATG initiationcodon and the ribosomal binding site of the B. subtilis gsiB gene andcloned in parent plasmid pBB1375 to create pBB1427.

Competent cells of B. subtilis strain 168 were prepared by the two-steptransformation method (Dubnau et al., 1994, Res. Microbiol. 145(5-6):403-411). The plasmid pBB1427 was used to transform the competent cells(of genotype ΔthyA Δ thyB sacA::[thyA⁺ cat]) to neomycin-resistance.Transformants arose by double-crossover recombination, resulting in theinsertion of the Pspac-tetC construct within the sacA locus. Arepresentative clone carrying genetic information for expression of TetCpeptide cytoplasmically was named BB2646. A control strain, BB2643,carrying the Pspac promoter at the sacA locus without the appended tetCcoding sequence was also prepared. This strain is a negative controlthat lacks genetic information encoding any antigen, i.e., carries anempty vector.

A strain displaying TTFC on the surface of spores as a fusion proteinwith CotC, a spore coat protein, was constructed by introducing intopSac-Kan a 374-bp DNA fragment that includes the cotC promoter andcoding sequence fused in-frame at its C-terminus with a 3-alanine-codonlinker and the coding sequence of TTFC (residues 2581 to 4237 of thetetanus toxin gene). The resulting plasmid, pBB1367, was introduced intothe ΔthyA ΔthyB sacA::[thyA⁺ cat] B. subtilis recipient strain bytransformation as described above, leading to integration at the sacAlocus and resistance to neomycin. A resulting transformant was namedBB2645.

Strains carrying three integrated copies of the Pspac-tetC or cotC-tetCconstruct at different loci (sacA, thrC and amyE) in the B. subtilischromosome were constructed. These strains, BB3059 for Pspac-tetC andBB3184 for cotC-tetC, were constructed by methods analogous to those forthe single copy integrants. The CotC fusion causes expression of theantigen on a spore coat protein. Expression of antigens on the surfaceof spores, or on the surface of vegetative cells or cytoplasmically invegetative cells is illustrated in FIG. 21. Constructs are shown in FIG.22.

Example 9 Preparation of Vegetative Cells and Extraction of VegetativeCell Lysates

Vegetative B. subtilis cells of strains BB2643 and BB2646 were preparedfor use in immunization by growth at 37° C. in LB to an absorbance at600 nm (OD₆₀₀) of 0.8-1.0. For vegetative cell lysates, cells were grownto OD₆₀₀=1.5 in LB medium or defined medium, the cell suspension waswashed and lysed by sonication, and collected by high-speedcentrifugation. Proteins concentrations were measured using the PierceProtein Assay kit (Thermo Fisher Scientific, Rockford Ill.). For Westernblotting, proteins were subjected to SDS-PAGE and blotted onnitrocellulose membranes. After successive incubation of the membranewith rabbit polyclonal anti-tetanus toxin antibody (1:500) and goatanti-rabbit IgG conjugated with horseradish peroxidase (Pierce, 1:500),the protein bands were visualized by chemiluminescence (Pierce),following the manufacturer's instructions (FIG. 6).

Example 10 Immunofluorescence Microscopy

B. subtilis strains (BB2643 and BB2646) grown in LB medium were fixed insitu as described previously (Harry et al., 1995, J. Bacterial. 177:3386-3393). Cultures were vortexed to disrupt clumps of bacteria beforefixation. A 0.25-ml volume of bacterial culture was mixed withconcentrated fixative solution to give 2.4% (vol/vol) parafamialdehyde,0.04% (vol/vol) glutaraldehyde, and 30 mM Na—PO4 buffer (pH 7.5) and themixture was incubated for 10 min at room temperature (20-22° C.) andthen for 50 min on ice. The fixed bacteria were washed three times inPBS, pH 7.4, at room temperature and were resuspended in 100 μl of GTE(50 mM glucose, 20 mM Tris-HCl, pH 7.5, 10 mM EDTA). A freshly preparedlysozyme solution in GTE was added to a final concentration of 2 mg/ml.Samples (10 μl) were immediately distributed into wells of a multiwellmicroscope slide (ICN Biochemicals; Aurora, Ohio) that had been treatedwith 0.1% (wt/vol) poly-L-lysine (Sigma). After 4 min, the liquid wasaspirated from the wells, which were then allowed to dry completely. Theslides were immersed in methanol at −20° C. for 5 min and then at −20°C. in acetone for 30 s and allowed to dry. Ten of blocking solution, 2%bovine serum albumin (BSA) in PBS (BSA-PBS) was added to each well, andslides were incubated for 15 min at room temperature and washed ninemore times. Samples were incubated with polyclonal rabbit anti-tetanustoxin for 1 h at room temperature, washed three times, and wereincubated with anti-rabbit immunoglobulin G (IgG)-fluoresceinisothiocyanate (Southern Biotech; Birmingham, Ala.) for 1 h at roomtemperature. After three washings, the samples were observed andphotographed with a Zeiss fluorescence microscope fitted with a NikonDMX1200 digital camera, and data were analyzed with Lucia GF software.

Example 11 Immunization Regimens

Groups of five 6- to 8-week old female BALB/c mice were inoculated viathe intranasal route with vegetative cells of various B. subtilisstrains B. subtilis vegetative cells were cultured in LB broth for 4-6hr until the culture reached an OD₆₀₀ of 0.8 to 1.0 at 600 nm. Afterharvesting, the cell pellets were resuspended in an equal volume of PBS.Spores were harvested after 48-72 hr of incubation with shaking in DSmedium (Fouet et al., 1990). The spores were washed repeatedly withsterile deionized water, treated with lysozyme (1 mg/ml) and washedagain several times. Residual contamination by vegetative cells, asdetected by phase contrast microscopy, was 1% or less. Spores werestored in deionized water at 4° C.

Mice were inoculated intranasally with 20 μl of cell or spore suspensionper dose (10⁷ to 10⁹ vegetative cells or 10⁸ to 10⁹ spores per dose; 10μl per nare) on days 0, 2, 14, 16, 28, and 30 (6 inoculations) or 0, 14,and 28 (3 inoculations). For a positive control, mice were immunizedintramuscularly (i.m.) with 50 μl of a commercial DTaP vaccine adsorbed(triple vaccine for diphtheria, tetanus and pertussis; Tripedia®, SanofiPasteur Inc., Swiftwater, Pa., USA) on days 0, 14, and 28 (3inoculations). Blood samples from inoculated mice were acquired on days−1, 13, 27, and 41.

Example 12 Detection of TTFC-Specific Serum Antibody Responses

Anti-TTFC antibody amount in serial three-fold dilutions of sera wasmeasured by ELISA. Absorbance values of pre-immune sera were used asreference blanks. Dilution curves were drawn for each serum sample andendpoint titers, representing the reciprocal value of the last dilutionthat gave an optical density ≧0.1, were expressed as the means±S.E. foranimals submitted to the same vaccine regimen. Serum TTFC-specific IgGsubclass responses were measured with same experimental procedure butusing peroxidase-conjugated rabbit anti-mouse IgG1 and IgG2a. Titerslower than 100 (negative samples) were arbitrarily assigned as 33.

Example 13 Tetanus Toxin Challenge

Three weeks following the last immunization, mice were challengedintraperitoneally with purified tetanus toxin (0.8 ng), determinedpreviously to be an amount that is twice LD₁₀₀. Mice were observed formorbidity or mortality daily for 10 days.

Example 14 Recombinant TTFC Expressed in B. Subtilis Vegetative Cells

A recombinant strain of B. subtilis was constructed to express the heavychain C fragment of tetanus toxin (TTFC), corresponding to the 457C-terminal amino acids of the 1315-residue tetanus holotoxin, from astrong and constitutively active mutant version of the spat promoter.This construct was integrated at the sacA locus in strain BB2646. TTFCexpression in BB2646 was confirmed by Western blotting andimmunofluorescent (IF) staining (FIG. 6).

Example 15 Oral Immunization with BB2646 Spores

Ability of spore preparations of strain BB2646 to generate a protectiveimmune response after oral immunization of mice was tested. Miceimmunized with the spore preparations showed very little increase inanti-TTFC serum antibody titer even after six inoculations with morethan 10¹⁰ spores per inoculation, compared to the control strain BB2643(FIG. 7). Constructs in which the TTFC-encoding sequence was fused to avegetative cell wall protein (WapA) or a spore coat protein (CotC) werealso tested. In neither of the latter cases was any significant increasein anti-TTFC titers in serum observed. Although some colonization of themouse GI tract by the recombinant strain could be detected, the BB2646titer in fecal samples declined within 7 days.

Example 16 Intranasal Immunization with BB2646 Spores

Ability of the BB2646 spore preparations from cells expressing theantigen cytoplasmically to immunize mice after intranasal inoculationwas tested. In this case, very high levels of serum anti-TTFC antibodieswere detected after three rounds of inoculation (one or two doses perround) at two-week intervals (FIG. 8). The titer after the third roundof immunization was the same whether the mice received a total of sixinoculations or three (FIG. 8) and was also as high as that generated byintramuscular inoculation with commercial DTaP vaccine.

These mice were completely protected from lethal toxin challenge (FIG.8). Co-administration of cholera toxin (CT) as an adjuvant did notaffect the observed immune response (FIG. 8). Mice inoculated withcontrol spores (strain BB2643) that were isogenic to BB2646 and lackedthe TTFC coding sequence gave no detectable antibody response and werefully sensitive to challenge by tetanus toxin (FIG. 8). The dose ofspores between 3×10⁸ and 3×10⁹ per dose was observed to give protectiveimmunity (FIG. 9).

Example 17 Mechanism of Intranasal Immunization by BB2646 Spores

Without being limited by any particular theory or mechanism of action, amodel the protective immunity afforded by spore preparations of BB2646might be due to germination of the spores in the nasopharynx, followedby outgrowth of vegetative cells, expression of TTFC and presentation ofthe TTFC to cells of the nasopharyngeal immune system. Howeverdissection of the nasopharynx of inoculated mice revealed the presenceof spores but not of any detectable level of vegetative cells. Moreover,incubation of the spores at 80° C. for 10 min or at 37° C. for 5 weeks,conditions which do not affect spore viability, greatly reducedimmunogenicity of the spore preparation (FIG. 10). In addition,introduction into strain BB2646 of a mutation that greatly reduced theability of the spores to germinate had only a small effect onimmunogenicity (FIG. 11). Finally, purification of the spores by densitygradient centrifugation removed the ability to induce an immune response(data not shown). Taken together these results suggest strongly that thespore form of strain BB2646 was not responsible for generating theprotective immunity that we had seen.

Example 18 Immunization by Vegetative Cells of BB2646

During spore preparation, contaminating vegetative cells were removed byosmotic shock, treatment with lysozyme and extensive washing to theextent that the level of contamination was no higher than 1% as measuredby phase contrast microscopy. Nonetheless, a low level of contaminationcould have been present. Since it is the vegetative form of BB2646 thatexpresses the TTFC antigen, whether such vegetative cells could accountfor the immunization obtained with spore preparations.

In fact, freshly grown vegetative cells harvested from growth medium andresuspended in PBS were observed to be very active inducers ofprotective immunity. Three doses of vegetative cells of BB2646 withtiters as low as 10⁷ cells per dose were observed to give a strongantibody response and protection against tetanus toxin (FIG. 12). Thus,vegetative cells contaminating the spore preparation could explainprotective immunity observed herein.

To explore the timing of development of immunogenic vegetative cellsfrom a population of spores, spores of stain BB2646 were heated to 80°C. for 10 min to kill any contaminating vegetative cells and the heatedspores were then suspended in LB and incubated at 37° C. At timedintervals samples of the germinating spores were removed and tested forimmunogenicity. The data show that unheated spore preparation gave astrong immune response at a dose of 10⁹; heating destroyedimmunogenicity (FIG. 13). After 1 hr of incubation at 37° C., an amountof culture equivalent to 10⁹ original spores was highly immunogenic(FIG. 13); microscopic examination revealed that these spores had losttheir refractility but had not yet grown out as vegetative cells. After3 hr in LB, the spore population had been converted almost entirely tovegetative cells. A sample corresponding to 10⁹ original sporesgenerated very high levels of serum antibody and full protection againsta tetanus toxin challenge (FIG. 13).

Example 19 Heat Stability of TTFC Expressed in B. Subtilis VegetativeCells

The rationale for using B. subtilis as a vaccine delivery system is thatthe spore form of the bacterium is highly resistant to a variety ofenvironmental conditions, including high temperatures, to whichconventional vaccines would be very sensitive. Since the active form ofthe vaccine strain engineered herein was observed to be the vegetativecell rather than the spore, ability of such vaccine strains to survivestorage at elevated temperatures was determined. Resistance to hightemperatures is particularly important for vaccine distribution andadministration in areas of the world that lack consistent and widespreadrefrigeration.

To evaluate antigenic stability to heating, B. subtilis vegetative cellswere incubated at 60° C. for 1 hr in either the wet state (in PBS) orafter drying in a Speed-Vac or freeze-drying in a lyophilizer. In thelatter cases, cells were resuspended in sterile H₂O after heating.

Mice that were immunized with vegetative cells that had been heated to60° C. in the wet state showed no increase in serum anti-TetC titers andwere indistinguishable from mice inoculated with control cells that donot express TTFC (FIG. 14 panel A). When the cells were heated in thedry state, however, very strong immune reactions were generated similarto those obtained with fresh, unheated vegetative cells, demonstratingthat the TTFC in dried vegetative cells was still highly immunogenicafter heat treatment. The mice immunized with cells that were heated inthe dry state were completely protected against lethal tetanus toxinchallenge (FIG. 14 panel B).

Similar studies were carried out with strain BB2645 that displays theTTFC on the surface of spores by fusion to the spore coat protein CotC.The immunogenicity of dried spores of this strain was entirely resistantto incubation at 60° C. for 60 min (FIG. 15).

To assess long-term heat stability at a temperature that is near thelimit of ambient conditions anywhere in the inhabited world, thesurvival of immunogenicity in dried preparations of vegetative cells andspores kept at 45° C. for 30 days was tested. For this example, strainsthat carried three copies of the Pspac-tetC (BB3059) or cotC-tetC(BB3184) construct were used to increase overall antigen delivery. Inboth cases, dried cells or spores were completely resistant to hightemperature, generating very high serum antibody responses at doses of10⁷-10⁸ per round of inoculation (FIGS. 16 and 17).

Example 20 Recombinant B. Subtilis Vegetative Cells Induced a BalancedTh1 and Th2 Immune Response

Ratios of IgG2a and IgG1 subclasses in host serum indicate the biastowards a Th1 or Th2 type immune response. Mice inoculated intranasallywith recombinant B. subtilis vegetative cells showed increased levels ofboth IgG1 and IgG2a, giving ratios near unity, whereas the micereceiving the conventional DTaP vaccine given i.m. had increased levelsof IgG1 but not of IgG2a, indicative of a Th-2 type immune response(FIG. 18). These results indicate that recombinant B. subtilisvegetative cells induced a balanced immune response.

Example 21 Recombinant B. Subtilis Spores Induced Increased IgA Levels

Mice were immunized intranasally with spores of strain BB2666, whichexpressed bovine VP6 under the control of spat promoter, or the controlstrain BB2643. See Examples 1 and FIGS. 1-4, and 21. Fecal samples werecollected two-weeks after the third round of inoculation and assayed forIgA-type antibodies by ELISA. Mice inoculated with rotavirus vaccinespores showed increased IgA level compared to mice inoculated withcontrol spores (FIGS. 19 and 20).

Example 22 Temperature Stability of B. Subtilis Spores

To test the heat stability of lyophilized B. subtilis spores stored fora period of time, mice were administered intranasally or sublinguallywith spores and the serum titer was determined.

Lyophilized B. subtilis spores BB3184 expressing TetC were lyophilizedand stored at 4° C. or 45° C. for 30 days, 90 days, and twelve months.Mice were immunized and serum samples were collected and analyzed forthe serum titer. FIG. 23 panels A and B show that the lyophilized B.subtilis spores stored at 4° C. or 45° C. for 30 days and 90 daysrespectively, produced high serum titer in animals. FIG. 24 shows thatlyophilized B. subtilis spores BB3184 stored at 4° C. or 45° C. fortwelve months also induced antibody production in animals. Data showthat 10⁹ lyophilized B. subtilis spores heated at 45° C. for either 30days or 90 days induced higher serum titer compared to 10⁸ cells oflyophilized B. subtilis spores incubated at 45° C. for the same amountof time.

FIG. 25 shows 100% survival rates for subjects immunized with 10⁹lyophilized B. subtilis spores heated at 45° C. for twelve months or at4° C. for twelve months. The survival rates for subjects immunized with10⁸ B. subtilis spores heated at 45° C. for twelve months was 60% threedays after challenge, and the survival rate was 40% seven days afterchallenge Animals immunized with control lyophilized spores died threedays after challenge. Thus, the lyophilized B. subtilis spores BB3184expressing TetC produced antibody production in subjects and weretemperature stable vaccines for at least twelve months.

Example 23 Temperature Stability of B. Subtilis Vegetative Cells

To test the stability of lyophilized B. subtilis vegetative cellsexpressing TetC, strains were prepared using recombinant methods. FIG.26 shows that TetC antigen is detectable as a soluble protein in themedia of vegetative cells of strain BB3059 which expresses TetC underthe control of Pspac promoter. TetC antigen represents about 3% of totalsoluble protein in vegetative cells BB3059. FIG. 27 shows a Western blotand Coomassie blue-stained gel showing expression of TetC antigen ineach of a recombinant B. subtilis vegetative cell strain producing toxinantigen under regulation of the IPTG-inducible Pspac, and in B. subtilisspores as fusion cotC-TetC. These vegetative cells and spores wereincubated for at least five hours at 37° C. Thus, lyophilized B.subtilis spores BB3184 and lyophilized B. subtilis vegetative cellsBB3059 display or express antigen that can be detected by assays.

Lyophilized B. subtilis vegetative cells BB3059 expressing TetCcytoplasmically and control lyophilized B. subtilis vegetative cells BB2643 were also lyophilized and stored at either 4° C. or 45° C. FIG. 28panels A and B show that lyophilized B. subtilis vegetative cellsexpressing TetC stored at 4° C. and 45° C. for 90 days induced highserum titer in murine subjects and provided protective immunogenicity tothe subjects. FIG. 29 panel A shows that lyophilized B. subtilisvegetative cells stored at 4° C. or 45° C. for twelve months inducedcomparable serum titer in the subjects. These figures show thatincreased concentration of the lyophilized vegetative cells expressingTetC increased the extent of protective immunogenicity.

FIG. 29 panel B shows 100% survival for murine subjects inoculated withlyophilized B. subtilis vegetative cells BB3059 expressing TetC,including those vegetative cells expressing TetC and stored at 4° C. or45° C. for 12 months. FIG. 29 panel B also shows that subjects immunizedwith control vegetative cells died three days after challenge.

To further analyze the protective immunogenicity of dried lyophilized B.subtilis vegetative cells BB3059 expressing TetC, cells were stored at45° C. for 17 months and used to immunize subjects. Groups of mice wereimmunized (2×10⁸ cells per dose) either intranasally with thelyophilized B. subtilis vegetative cells BB3059, sublingually withlyophilized B. subtilis vegetative cells BB3059, or intranasally withcontrol lyophilized B. subtilis vegetative cells BB2643 lacking the TetCcoding sequence. Sublingual administration involved sedating thesubjects then placing 20 μL of the material under the tongue of eachsubject. This sublingual administration procedure was used in subsequentexamples unless otherwise indicated.

FIG. 30 panel A shows the average amount of serum anti-TetC antibodiesdetected in the subjects as a function of time (days) after theimmunization. Subjects inoculated intranasally with lyophilized B.subtilis vegetative cells expressing TetC and mice inoculatedsublingually with lyophilized B. subtilis vegetative cells expressingTetC showed orders of magnitude greater serum antibody titer than miceintranasally immunized with control lyophilized B. subtilis vegetativecells BB2643 (FIG. 30 panel A).

Ability of the lyophilized BB3059 vegetative cells to immunizeindividual mice after intranasal or sublingual inoculation was alsodetermined and is shown in FIG. 30 panel B. Higher levels of serumanti-TetC antibodies were detected in individual mice intranasally orsublingually immunized with lyophilized B. subtilis vegetative cellsBB3059 compared to mice immunized with control cells.

Thus, dried lyophilized B. subtilis vegetative cells BB3059 expressingTetC were heat-stable and effective vaccines.

Example 24 Analysis of Lyophilized B. Subtilis Spores and VegetativeCells

FIG. 31 is a chart showing the safety and vaccination steps used fornormal mice and SCID in an analysis performed to determine theeffectiveness of the vaccination spores and vegetative cells. Five micewere terminated three days after every immunization and their olfactorylobes, cerebrum, lung, and nasal epithelium including thenasal-associated lymphoid tissue (NALT) were examined. Immunizedsubjects were found to have normal tissue morphology indicative ofprotective immunogenicity. Subject body weight was also monitored dailyas shown in FIG. 32.

Data show that vaccines were heat stable and fully protective andprovided systemic immunity against tetanus when administeredintranasally or mucosally. Serum antibody titer values were greater than30,000 which correlated with 100% protection against two times thelethal amount of tetanus toxin. Serum titer of greater than 10,000correlating generally with about 80% protection. Further analysis wasperformed on the vaccines to further optimize the methods andcompositions shown herein.

Example 25 Sublingual Administration of Lyophilized B. SubtilisVegetative Cells with mLT Adjuvant

To evaluate the effect of mLT adjuvant on serum titer and survivalrates, mice were sublingually immunized with lyophilized B. subtilisvegetative cells BB3059 expressing cotC-TetC (with or without adjuvant)and control vegetative cells BB2643.

Serum samples were collected after inoculation and assayed by ELISA forserum titer. FIG. 33 panel A shows that regardless of presence of mLTadjuvant, mice inoculated with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC showed higher serum titer compared to miceinoculated with control vegetative cells. FIG. 33 panel B shows 100%survival for murine subjects sublingually immunized with lyophilized B.subtilis vegetative cells BB3059 expressing TetC, compared to 40% forsubjects sublingually immunized with lyophilized. B. subtilis vegetativecells BB3059 expressing TetC and mLT adjuvant. Subjects sublinguallyimmunized control lyophilized B. subtilis vegetative cells BB2643 diedthree days after challenge.

Example 26 Sublingual Administration of Lyophilized B. SubtilisVegetative Cells and Purified Recombinant TetC

Mice were sublingually immunized with lyophilized B. subtilis BB3059expressing TetC (with or without adjuvant), a purified recombinant TetCfragment or a control vegetative cells BB2643 to compare serum titer andsurvival rates.

FIG. 34 panel A shows comparable serum titer values for subjectsimmunized intranasally with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC, subjects immunized sublingually with lyophilizedB. subtilis vegetative cells BB3059 expressing TetC, and subjectsimmunized sublingually with recombinant TetC fragment.

Data in FIG. 34 panel B shows 100% survival for tetanus challengedsubjects immunized either intranasally or sublingually with lyophilizedB. subtilis vegetative cells BB3059 expressing TetC. Subjects immunizedsublingually with purified recombinant TetC fragment were observed tohave 80% survival three days after immunization. Subjects immunized withcontrol vegetative cells died three days after challenge.

Example 27 Lyophilized B. Subtilis Vegetative Cells Expressing TetCInduced a Balanced Th1 and Th2 Immune Response

To further investigate the effect administering lyophilized B. subtilisvegetative cells expressing TetC has on the immune response, serum fromimmunized mice were analyzed for IgG1 and IgG2a. Subjects were immunizedsublingually with lyophilized B. subtilis vegetative cells BB3059expressing TetC; intranasally with lyophilized B. subtilis vegetativecells BB3059 expressing TetC; sublingually with purified recombinantTetC fragment; or intramuscularly with a commercial vaccine fordiphtheria, tetanus and pertussis (DTAP; Tripedia®, Sanofi Pasteur Inc.,Swiftwater, Pa., USA).

FIG. 35 shows that mice inoculated intranasally or sublingually withrecombinant lyophilized B. subtilis vegetative cells BB3059 wereobserved to have very similar levels of IgG1 and IgG2a. The subjectsinoculated sublingually with strain BB3059 had a ratio of IgG1 and IgG2aof 18, and subjects inoculated intranasally with strain BB3059 had aratio of IgG1 and IgG2a of 44.6. The ratio of IgG1 and IgG2a was muchmore disproportionate for the subjects immunized sublingually withrecombinant TetC fragment (160.2) and intramuscularly with the DTaPvaccine (5532.6). The increased ratio of IgG1 compared to IgG2a isindicative of a disproportionate Th-2 type immune response instead of abalanced Th-1 and Th-2 response. Thus data show that intranasally andsublingually administered B. subtilis vegetative cells expressing TetCinduced a more balanced Th-1 and Th-2 immune response than a recombinantTetC fragment and a commercially available DTAP vaccine.

Example 28 Cytokines Induced by Lyophilized B. Subtilis Vegetative CellsExpressing TetC

Serum cytokines induced by inoculation with lyophilized B. subtilisvegetative cells BB3059 expressing TetC were also analyzed and comparedto results for commercially available DTaP vaccine.

FIG. 36 panels A-D show serum cytokine serum levels two weeks after athird immunization of subjects. The mice were immunized sublinguallywith lyophilized B. subtilis vegetative cells BB3059, intranasally withlyophilized B. subtilis vegetative cells BB3059, sublingually withpurified recombinant TetC fragment, or intramuscularly with commercialDTAP vaccine.

FIG. 36 panel A shows that subjects sublingually or intranasallyimmunized with lyophilized B. subtilis vegetative cells BB3059 had twiceas much interleukin-2 in serum compared to subjects immunizedsublingually with either purified recombinant TetC and orintramuscularly with DTAP vaccine. FIG. 34 panel B shows substantialinterferon-gamma serum concentrations for subjects sublinguallyimmunized with lyophilized B. subtilis vegetative cells BB3059expressing TetC and subjects immunized with intranasally withlyophilized B. subtilis vegetative cells BB3059 expressing TetC,sublingually with purified recombinant TetC, or intramuscularly withDTAP vaccine. FIG. 34 panel C shows increased interleukin-4 serumconcentrations for subjects intranasally immunized with lyophilized B.subtilis vegetative cells BB3059 expressing TetC. FIG. 34 panel D showscomparable interleukin-10 serum concentrations for subjects immunizedsublingually with purified recombinant TetC compared to subjectssublingually or intranasally immunized with lyophilized B. subtilisvegetative cells BB3059, or subjects immunized sublingually with DTAPvaccine. Thus, lyophilized B. subtilis vegetative cells BB 3059expressing TetC induced greater or comparable amounts of serumcytokines.

Example 29 Lyophilized B. Subtilis Vegetative Cells Expressing TetCIncreased Immunoglobulin Levels

Fecal, vaginal and saliva anti-TetC IgG and IgA levels were analyzed inmice inoculated with lyophilized B. subtilis vegetative cells expressingTetC. Mice were immunized: sublingually with control lyophilizedvegetative cells, sublingually with lyophilized B. subtilis vegetativecells BB3059 expressing TetC, intranasally with lyophilized B. subtilisvegetative cells BB3059 expressing TetC, or intramuscularly withcommercial DTAP vaccine.

FIG. 37 panel A shows high IgG fecal content in mice inoculatedsublingually with lyophilized B. subtilis vegetative cells BB3059expressing TetC, subjects inoculated intramuscularly with commercialDTAP vaccine, and subjects inoculated intranasally with lyophilized B.subtilis vegetative cells BB3059 expressing TetC. Little or no IgG fecalcontent was observed in subjects inoculated sublingually with controllyophilized B. subtilis vegetative cells. FIG. 37 panel B shows high IgAfecal content in mice inoculated intranasally with lyophilized B.subtilis vegetative cells BB3059 expressing TetC and mice inoculatedsublingually with B. subtilis vegetative cells BB3059 expressing TetC.Little or no IgA fecal content was observed in mice inoculatedsublingually with control lyophilized B. subtilis vegetative cells ormice inoculated intramuscularly with commercial DTAP vaccine.

Subjects were immunized sublingually with lyophilized B. subtilisvegetative cells BB3059 expressing TetC, intranasally with lyophilizedB. subtilis vegetative cells BB3059 expressing TetC, sublingually withpurified recombinant TetC, or intramuscularly with commercial DTAPvaccine. Murine vaginal samples and saliva samples were assayed forTetC-specific IgA. FIG. 38 shows that higher amounts of TetC-specificIgA were detected in the vaginal samples compared to the saliva samples.In both the vaginal samples and saliva samples, higher amounts ofTetC-specific IgA were detected in subjects immunized sublingually withlyophilized B. subtilis vegetative cells BB3059 expressing TetC, and thesubjects immunized intranasally with lyophilized B. subtilis vegetativecells BB3059 expressing TetC. Much lower levels of TetC-specific IgAwere detected in subjects immunized sublingually with purifiedrecombinant TetC (30%-60% less) and in subjects immunizedintramuscularly with commercial DTAP vaccine (90% less).

Thus, lyophilized B. subtilis vegetative cells expressing TetC generallyresulted in greater immunoglobulin amounts in excretions or body fluidsthan results for commercial DTaP vaccine.

Example 30 Lyophilized B. Subtilis Vegetative Cells Expressing TetCIncreased Immunoglobulin Levels and Reduced MHC Class II Staining

MHC class II staining of tissues of mice inoculated with B. subtilisvegetative cells expressing TetC compared to tissue inoculated withcontrol vegetative cells was analyzed and compared to commercialvaccines. Mice were immunized sublingually with either lyophilized B.subtilis vegetative cells BB3059 expressing TetC or control vegetativecells.

Subjects were sacrificed 24 hours after sublingual immunization and MHCclass II deposition staining was performed on the murine spleen tissue(FIG. 39 panels A and B) and murine intestinal tract tissue (FIG. 39panels C and D).

FIG. 39 shows extensive MHC class II deposition on murine spleen tissue(panel A) and murine intestinal tract tissue (panel C), respectively,from mice sublingually immunized with lyophilized B. subtilis vegetativecells BB3059 expressing TetC. Extensive MAC immunostaining with variouspatterns of staining of the cells was observed. FIG. 39 panels B and Dshow MHC class II deposition on murine spleen tissue and murineintestinal tract tissue, respectively, from mice sublingually immunizedwith control vegetative cells. Little or no MHC class II immunostainingwas observed on the murine tissue.

Thus, mice inoculated with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC showed much more MHC class II deposition comparedto mice inoculated with control cells.

Example 33 Inoculation Using Adjuvant and Lyophilized B. SubtilisVegetative Cells

Fecal, serum and saliva samples from piglets inoculated with lyophilizedB. subtilis vegetative cells BB3059 expressing TetC were analyzed forimmunoglobulin levels.

Anti-mLT IgG and IgA levels were detected in serum, fecal and salivasamples from piglets 14 days after a fourth sublingual immunization withlyophilized B. subtilis vegetative cells expressing TetC with adjuvantor without mLT adjuvant. The data in FIG. 40 show that in each of theserum, fecal, and saliva samples animals administered lyophilized B.subtilis lyophilized B. subtilis vegetative cells BB3059 with mLTadjuvant produced higher levels of IgG and IgA than animals administeredlyophilized control vegetative cells only.

FIG. 41 shows a serum and fecal anti-mLT IgG and IgA titer for mice 14days after a third immunization or fourth immunization. The subjectswere immunized either sublingually with lyophilized B. subtilisvegetative cells expressing TetC and 5 μg of mLT adjuvant (LT 5+vegcells, SL; closed bars), or intranasally with lyophilized B. subtilisvegetative cells expressing TetC and 10 μg of mLT adjuvant (LT 10+vegcells, IN; open bars). FIG. 41 panel A shows comparable serum anti-mLTIgG and IgA for mice sublingually and intranasally immunized. FIG. 41panel B shows that fecal IgA was higher than fecal IgG for bothsublingually or intranasally immunized subjects.

Example 31 Inoculation Using Adjuvant and Lyophilized B. SubtilisVegetative Cells Expressing TetC

Piglets were immunized sublingually with lyophilized B. subtilisvegetative cells BB3059 expressing TetC, sublingually with lyophilizedB. subtilis vegetative cells BB3059 expressing TetC and mLT adjuvant,orally with lyophilized B. subtilis vegetative cells BB3059 expressingTetC, and sublingually with control lyophilized B. subtilis vegetativecells. Serum samples were collected and assayed for TetC specificantibodies.

FIG. 42 shows higher serum anti-TetC antibody titer for subjectsimmunized sublingually with lyophilized B. subtilis vegetative cellsBB3059 expressing TetC with or without mLT adjuvant compared to subjectsimmunized orally with either lyophilized B. subtilis vegetative cellsBB3059 expressing TetC or control vegetative cells. No statisticallysignificant difference was observed in immunoglobulin levels betweeninoculating subjects with strain BB3059 expressing TetC with or withoutmLT. Control vegetative cells did not induce antibody production insubjects.

Example 32 Optimization of Immunization Schedule

To further determine the optimal immunization schedule, subjects wereimmunized at different intervals and serum titer was analyzed.

FIG. 43 shows serum titer (ordinate) as a function of time (abscissa) inmice immunized with dried vegetative cell vaccine expressing TetCaccording to the following schedule: biweekly, monthly, or bimonthly.The data show high serum titer for mice immunized biweekly and monthly.

Example 33 Vaccination Against Ragweed Using Recombinant Lyophilized B.Subtilis Spores and Vegetative Cells

Compositions of lyophilized B. subtilis spores and vegetative cells areprepared that encode and express an antigen known to be an allergen, forexample, the whole of or a part of antigen E of ragweed (genus Ambrosiaor other members of Asteraceae), which is an antigen associated withthis pollen.

Spores and vegetative cells are prepared that encode all or a portion ofantigen E of ragweed either displaying on the surface of the spore orproduced cytoplasmically in the cell. The constructs are grown andproduction of the antigen is determined by Western blot or immunoassays.

Subjects are inoculated with the lyophilized B. subtilis spores orlyophilized B. subtilis vegetative cells carrying the constructsencoding the ragweed antigen. Serum titer, extent of protectiveimmunogenicity, minimum effective dose, and survival rates aredetermined, and data are obtained to show that the constructs arepotential improved vaccination agents. The constructs are tested usingnumerous routes of administration, for example sublingual, intranasal,oral, ocular, rectal, vaginal, and intravenous.

Immunological data are obtained to determine the extent that thelyophilized B. subtilis spores and vegetative cells protect cells,tissue and a subject from developing indicia of allergic reactions, orreduce the severity of the allergic reaction to ragweed.

Advantages of inoculating a subject with the lyophilized B. subtilisspores and vegetative cells encoding a ragweed antigen are shown.Improved immunization results are achieved with these constructscompared to standard methods of treating allergies including ingestingprescription drugs for example antihistamines and steroids, or areceiving a regimen of allergy shots that block the effects of IgEantibodies. The lyophilized B. subtilis spores and lyophilized B.subtilis vegetative cells individually or in combination are also shownto be effective.

Sublingual or intranasal immunization schedules can be carried out bythe patient using prepared unit dosage kits, greatly reducing oreliminating health care costs for allergy patients. Methods,compositions and kits herein are made for other allergens, for exampledust mite proteases and gluten.

Example 34 Vaccination Against Tumor Antigens Using RecombinantLyophilized B. subtilis Spores and Vegetative Cells

Lyophilized B. subtilis spores and vegetative cells containingrecombinant constructs encoding and expressing an antigen present ontumor or a protein associated with the presence or growth of a tumor orcancer are prepared. The antigen of the tumor is for example humanepidermal growth factor receptor 2 (HER2) which is associated with aclass of breast cancer tumors. Such immunization is particularlysuitable for breast cancer survivors in remission, to prevent arecurrence and metastasis.

Subjects are administered lyophilized B. subtilis spore or vegetativecell vaccine prepared from cells having constructs encoding the HER2antigen. The constructs are tested using numerous routes ofadministration, for example sublingual, intranasal, oral, ocular,rectal, vaginal, and intravenous.

Data show that administering the lyophilized B. subtilis spores andvegetative cells encoding the HER2 antigen protects the subject from theindicia of breast cancer tumors. The lyophilized B. subtilis spores andlyophilized B. subtilis vegetative cells shown herein are also usedunder different conditions or administration methods, or in combination.

Numerous other lyophilized B. subtilis spores and vegetative cellscontaining recombinant constructs encoding and expressing an antigen areprepared, for example the vaccination antigen is for example nestin,carcinoembryonic antigen (CEA), cancer antigen 125 (CA-125) and humanchorionic gonadotropin (HCG).

What is claimed is:
 1. A method of using a heat stable vaccinepreparation to immunize a subject to an infectious agent, a tumor, or anallergen, the method comprising: providing a Bacillus subtilis bacterialcell culture containing Bacillus subtilis vegetative cells or providinga preparation of Bacillus subtilis spores, the vegetative cells and thespores each expressing an isolated nucleotide sequence encoding anantigen of the infectious agent, the tumor, or the allergen, thenucleotide sequence is operably linked to either: a vegetative promoterfor cytoplasmic vegetative expression of the antigen in the vegetativecells, or a sporulation promoter for expression of the antigen as afusion to a spore coat protein on the Bacillus subtilis spores;collecting the cells or the spores from the culture by centrifugationand heat-stabilizing the antigen by lyophilizing the vegetative cells orthe spores to obtain a heat stable vaccine preparation having heatstable immunogenic potency for at least 12 months at a temperature of45° C. comparable to the vaccine stored at 4° C.; and, contacting amucosal tissue of the subject with the heat stable vaccine preparationwhich induces an immune response and immunizes the subject to theinfectious agent, the tumor, or to the allergen.
 2. The method accordingto claim 1, wherein the infectious agent is selected from the group of:a bacterium, a fungus, a virus, a protozoan, or a protein productthereof.
 3. The method according to claim 2, wherein the infectiousagent is at least one bacterium selected from the group consisting of:Bacillus anthracis; Clostridium tetani; Clostridium difficile;Clostridium perfringens; Corynebacterium diphtheriae; Bordetellapertussis; Mycobacterium tuberculosis; Salmonella enterica;Staphylococcus aureus; Staphylococcus epidermis; Streptococcuspneumoniae; Streptococcus mutans; Treponema pallidum; Pseudomonasaeruginosa; Neisseria gonorrhoeae; Escherichia coli; Escherichia coliO157:H7; Shigella enteritis; Shigella flexneri; Campylobacter jejuni;Yersinia pseudotuberculosis; Yersinia pestis; Listeria monocytogenes;and Vibrio cholerae.
 4. The method according to claim 2, wherein theinfectious agent is at least one virus selected from the groupconsisting of: human immunodeficiency virus (HIV); influenza virus A;influenza virus B; influenza virus A/H1N1; polio; Herpes simplexvirus-1; Herpes simplex virus-2; smallpox; measles; mumps; rubella;rotavirus; chicken pox; rabies; West Nile virus; Ebola hemorrhagicfever; eastern equine encephalitis; norovirus; Hepatitis A; Hepatitis B;and Hepatitis C.
 5. The method according to claim 2, wherein theinfectious agent is at least one fungus selected from the groupconsisting of: Cryptococcus Gattii: Cryptococcus neoformans v.neoformans; Candida albicans; Aspergillus flavus; and Aspergillusfumigatus.
 6. The method according to claim 2, wherein the infectiousagent is at least one protozoan selected from the group consisting of:Entamoeba histolytica; Giardia lamblia; Cryptosporidium parvum;Naegleria fowleri; Naegleria gruberi; Plasmodium falciparum; Plasmodiumvivax; Plasmodium malariae; and Plasmodium ovale.
 7. The methodaccording to claim 1, wherein the antigen is a rotavirus antigenselected from the group of: bovine, human, and murine origin.
 8. Themethod according to claim 7, wherein the rotavirus antigen is a viralvirion protein selected from at least one of the group of VP2, VP4, VP6,VP7, NSP4, and a portion or a derivative thereof.
 9. The methodaccording to claim 1, wherein the allergen comprises a macromolecule orportion thereof associated with an increased immunoglobulin level orallergic response in the subject, wherein the allergen comprises anenvironmental allergen, animal or plant allergen, or food allergen,selected from the group consisting of: allergen associated with pollen,dust mite proteases, fungus, mold, pet dander, saliva, shellfish,seafood, a legume, and peanuts.
 10. The method according to claim 1,wherein the subject is a vertebrate animal.
 11. The method according toclaim 10, wherein the vertebrate animal is selected from at least one ofthe group consisting of: an agricultural animal, a high value zooanimal, a research animal, a human, and a wild animal in a dense humanenvironment.
 12. The method according to claim 1, wherein contacting themucosal tissue of the subject further comprises administering thecomposition by a route selected from at least one of the group of:intravenous, intramuscular, intraperitoneal, intradermal,intrapulmonary, intravaginal, rectal, oral, buccal, sublingual,intranasal, intraocular, and subcutaneous.
 13. The method according toclaim 1, wherein contacting the mucosal tissue of the subject comprisesapplying to the mucosal tissue at least one from the group of: anaerosol, a mist, a nose drop, an eye drop, a mouth drop, a capsule, atablet, a pill, a powder, a granule, a fluid, a suspension, an emulsion,a gel, a patch, and a lozenge.
 14. The method according to claim 1,wherein contacting the mucosal tissue of the subject further comprisescontacting the mucosal tissue with an adjuvant.
 15. The method accordingto claim 14, wherein the adjuvant is selected from at least one of thegroup or: cholera toxin, a non-toxic variant of Escherichia coli labiletoxin, and a portion or a derivative thereof.
 16. The method accordingto claim 1, further comprising observing resistance of the compositionto at least one condition selected from the group of: heat, drying,freezing, deleterious chemicals, and radiation.
 17. The method accordingto claim 16, wherein observing the resistance to heat comprisesobserving resistance at 60° C. or 45° C. for at least one period of timeselected from the group of: at least one month, at least six months, atleast one year, and at least two years.
 18. The method according toclaim 16, wherein observing resistance comprises observing a heattreated composition maintaining ability to confer full protectiveimmunity or partial protective immunity, wherein the partial protectiveimmunity comprises a percentage of the full protective immunity, whereinthe percentage comprises at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90%.
 19. The method according toclaim 1, further comprising: measuring an antibody titer in scrum of thesubject administered the composition, wherein an increase in antibodyfor the antigen in comparison to a control serum is an indication ofefficacy of the immunogenicity of the composition.
 20. The methodaccording to claim 1, further comprising: measuring an amount of antigenshedding in the subject having been afflicted by the infectious agent,wherein a decrease in fecal antigen as compared to that in a controlalso afflicted by the infectious agent and not contacted with thecomposition is a measure of efficacy of the immunogenicity of thecomposition.
 21. The method according to claim 1, wherein the antigencomprises a Clostridium tetani toxin antigen, wherein the nucleotidesequence is operably linked to the promoter for the cytoplasmicvegetative expression of the Clostridium tetani toxin antigen or forexpression of the Clostridium tetani toxin antigen as the fusion to thespore coat protein, and the vegetative cell and the spore are associatedwith the Clostridium tetani toxin antigen.
 22. A method of using a heatstable vaccine preparation to immunize a subject to an infectious agent,a tumor, or an allergen, the method comprising: providing Bacillusvegetative cells expressing an isolated nucleotide sequence encoding anantigen or the infectious agent, the tumor, or the allergen, thenucleotide sequence is operably linked to a Pspac promoter containing asequence comprising SEQ ID NO: 1 for cytoplasmic vegetative expressionof the antigen; collecting the cells from the culture by centrifugation,and heat-stabilizing the antigen by lyophilizing the vegetative cells toobtain the resulting heat stable vaccine preparation; and, contacting amucosal tissue of the subject with the heat stable vaccine preparationwhich induces an immune response and immunizes the subject to theinfectious agent, the tumor, or to the allergen.
 23. A method of using aheat stable vaccine preparation to immunize a subject to an infectiousagent, a tumor, or an allergen, the method comprising: providingBacillus subtilis spores expressing an isolated nucleotide sequenceencoding an antigen of the infectious agent, the tumor, or the allergen,the nucleotide sequence is operably linked to a gene for a spore coatprotein for expression of the antigen as a fusion to the spore coatprotein; collecting the spores from the culture by centrifugation, andheat-stabilizing the antigen expressed on the spore coat protein bylyophilizing the spores to obtain the resulting heat stable vaccinepreparation which is stable for at least 12 months at a temperature of45° C. comparable to the vaccine stored at 4° C.; and, contacting amucosal tissue of the subject with the heat stable vaccine preparationwhich induces an immune response and immunizes the subject to theinfectious agent, the tumor, or to the allergen.